RESUMO
IMPORTANCE: Spacer oligonucleotide typing (spoligotyping), the first-line genotyping assay for Mycobacterium tuberculosis (MTB), plays a fundamental role in the investigation of its epidemiology and evolution. In this study, we established a single-tube spoligotyping assay using MeltArray, a highly multiplex polymerase chain reaction (PCR) approach that runs on a real-time PCR thermocycler. The MeltArray protocol included an internal positive control, gyrB, to indicate the abundance of MTB via the quantification cycle and 43 spacers to identify the spoligotype via melting curve analysis. The entire protocol was completed in a single step within 2.5 hours. The lowest detectable copy number for the tested strains was 20 copies/reaction and thus sufficient for analyzing both culture and sputum samples. We conclude that MeltArray-based spoligotyping could be used immediately in low- and middle-income countries with a high tuberculosis burden, given its easy access, improved throughput, and potential applicability to clinical samples.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Epidemiologia Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Multiplex , Técnicas de Tipagem Bacteriana/métodos , GenótipoRESUMO
OBJECTIVES: This study aimed to measure the prevalence of resistance to antimicrobial agents, and explore the risk factors associated with drug resistance by using nontuberculous Mycobacteria (NTM) isolates from China. METHODS: A total of 335 NTM isolates were included in our analysis. Broth dilution method was used to determine in vitro drug susceptibility of NTM isolates. RESULTS: Clarithromycin (CLA) was the most potent drug for Mycobacterium intracellulare (MI). The resistance rate of 244 MI isolates to CLA was 21%, yielding a minimum inhibitory concentrations (MIC)50 and MIC90 of 8 and 64 mg/L, respectively. 51% of 244 MI isolates exhibited resistance to amikacin (AMK). For 91 Mycobacterium abscessus complex (MABC) isolates, 6 (7%) and 49 (54%) isolates were categorized as resistant to CLA at day 3 and 14, respectively. The resistance rate to CLA for Mycobacterium abscessus subspecies abscessus (MAA) was dramatically higher than that for Mycobacterium abscessus subspecies massiliense (MAM). Additionally, the percentage of patients presenting fever in the CLA-susceptible group was significantly higher than that in the CLA-resistant group. CONCLUSIONS: Our data demonstrate that approximate one fifth of MI isolates are resistant to CLA. We have identified a higher proportion of CLA-resistant MAA isolates than MAM. The patients caused by CLA-resistant MI are at low risk for presenting with fever relative to CLA-susceptible group.
Assuntos
Mycobacterium abscessus , Micobactérias não Tuberculosas , Humanos , Complexo Mycobacterium avium , China , Amicacina , Claritromicina , FebreRESUMO
The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase , Mutação , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Proteínas de Bactérias/genéticaRESUMO
The diagnosis of multidrug-resistant tuberculosis (MDR-TB) is crucial for the subsequent drug guidance to improve therapy and control the spread of this infectious disease. Herein, we developed a novel florescence biosensor for simultaneous detection of Mycobacterium tuberculosis (Mtb) multidrug-resistant genes (rpoB531 for rifampicin and katG315 for isoniazid) by using our synthesized nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (nanoCoTPyP) and double quantum dots (QDs). Several nanoCoTPyPs with different charges and morphology were successfully prepared via the surfactant-assisted method and their quenching ability and restoring efficiency for DNA detection were systematically analyzed. It was found that spherical nanoCoTPyP with positive charge exhibited excellent quenching effect and sensing performance for the two DNAs' detection due to its affinity differences towards single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). ssDNA attached on QDs (QDs-ssDNA) was specifically hybridized with targets to form QDs-dsDNA, resulting in fluorescence recovery due to the disruption of the interactions between nanoCoTPyP and ssDNA. Two drug-resistant genes could be simultaneously quantified in a single run and relatively low limits of detection (LODs) were obtained (24 pM for T1 and 20 pM for T2). Furthermore, the accuracy and reliability of our method were verified by testing clinical samples. This simple and low-cost approach had great potential to be applied in clinical diagnosis of MDR-TB.
Assuntos
Técnicas Biossensoriais , Mycobacterium tuberculosis , Pontos Quânticos , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Técnicas Biossensoriais/métodos , DNA , DNA de Cadeia Simples , Humanos , Isoniazida , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
BACKGROUND: We performed a prospective multicentre diagnostic study to evaluate the combined interferon-γ (IFN-γ) and interleukin-2 (IL-2) release assay for detect active pulmonary tuberculosis (TB) in China. METHODS: Adult patients presenting symptoms suggestive of pulmonary TB were consecutively enrolled in three TB-specialized hospitals. Sputum specimens and blood sample and were collected from each participant at enrolment. The levels of Mycobacterium tuberculosis (MTB)-specific antigen-stimulated IFN-γ and IL-2 were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: Between July 2017 and December 2018, a total of 3245 patients with symptoms suggestive of pulmonary TB were included in final analysis. Of 3245 patients, 2536 were diagnosed as active TB, consisting of 1092 definite TB and 1444 clinically diagnosed TB. The overall sensitivity and specificity of IFN-γ were 83.8% and 81.5%, respectively. In addition, compared with IFN-γ, the specificity of IL-2 increased to 94.3%, while the sensitivity decreased to 72.6%. In addition, the highest sensitivity was achieved with parallel combination of IFN-γ/IL-2, with a sensitivity of 87.9%, and its overall specificity was 79.8%. The sensitivity of series combination test was 68.5%. Notably, the sensitivity of series combination test in definite TB (72.1%) was significantly higher than that in clinically diagnosed TB (65.8%). CONCLUSION: In conclusion, we develop a new immunological method that can differentiate between active TB and other pulmonary diseases. Our data demonstrates that the various IFN-γ/IL-2 combinations provides promising alternatives for diagnosing active TB cases in different settings. Additionally, the diagnostic accuracy of series combination correlates with severity of disease in our cohort.
Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar , Adulto , Antígenos de Bactérias , China , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama , Testes de Liberação de Interferon-gama , Interleucina-2 , Estudos Prospectivos , Tuberculose Pulmonar/diagnósticoRESUMO
BACKGROUND: The positive rate of pathogenic examination about tuberculosis is low. It is still difficult to achieve early diagnosis for some TB patients. The value of Interferon-gamma release assays (IGRA) in the diagnosis of active tuberculosis remains controversial. The purpose of this multicenter prospective study was to verify and validate the role of TBAg/PHA ratio (TB-specific antigen to phytohaemagglutinin) of T-SPOT.TB assay in diagnosing ATB. METHODS: We prospectively enrolled 2390 suspected pulmonary tuberculosis patients with positive T-SPOT assay results from three tertiary hospitals. RESULTS: A total of 1549 ATB (active tuberculosis) patients (including 1091 confirmed and 458 probable ATB) and 724 non-tuberculosis (non-TB) patients with positive T-SPOT results were included. The results of this study showed that ESAT-6 and CFP-10 in the T-SPOT.TB assay were significantly higher in the ATB group compared with the non-TB group, while PHA was lower in the ATB group. Results of ESAT-6, CFP-10 and PHA show a certain diagnostic performance, but moderate sensitivity and specificity. The TBAg/PHA ratio, a further calculation of ESAT-6, CFP-10 and PHA in T-SPOT.TB assay showed improved performance in the diagnosis of active Tuberculosis. If using the threshold value of 0.2004, the specificity and sensitivity of TBAg/PHA ratio in distinguishing ATB from non-TB were 92.3% and 74.4%, PPV was 95.4, PLR was 9.6. CONCLUSION: By recalculating the results of T-SPOT.TB Assay, the TBAg/PHA ratio shows high prospect value in the diagnosis of active tuberculosis in high prediction areas.
Assuntos
Antígenos de Bactérias/metabolismo , Mycobacterium tuberculosis/imunologia , Fito-Hemaglutininas/metabolismo , Tuberculose Pulmonar/diagnóstico , Antígenos de Bactérias/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Estudos Prospectivos , Reprodutibilidade dos Testes , Escarro/metabolismo , Escarro/microbiologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc1 inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.
Assuntos
Antibacterianos/farmacologia , Clofazimina/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium abscessus/efeitos dos fármacos , Amicacina/farmacologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Claritromicina/farmacologia , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Engenharia Genética/métodos , Luminescência , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/enzimologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Imagem Óptica/métodos , Recombinases/genética , Recombinases/metabolismo , Roxitromicina/farmacologiaRESUMO
BACKGROUND: Perturbed iron homeostasis is a risk factor for tuberculosis (TB) progression and an indicator of TB treatment failure and mortality. Few studies have evaluated iron homeostasis as a TB diagnostic biomarker. METHODS: We recruited participants with TB, latent TB infection (LTBI), cured TB (RxTB), pneumonia (PN) and healthy controls (HCs). We measured serum levels of three iron biomarkers including serum iron, ferritin and transferrin, then established and validated our prediction model. RESULTS: We observed and verified that the three iron biomarker levels correlated with patient status (TB, HC, LTBI, RxTB or PN) and with the degree of lung damage and bacillary load in patients with TB. We then built a TB prediction model, neural network (NNET), incorporating the data of the three iron biomarkers. The model showed good performance for diagnosis of TB, with 83% (95% CI 77 to 87) sensitivity and 86% (95% CI 83 to 89) specificity in the training data set (n=663) and 70% (95% CI 58 to 79) sensitivity and 92% (95% CI 86 to 96) specificity in the test data set (n=220). The area under the curves (AUCs) of the NNET model to discriminate TB from HC, LTBI, RxTB and PN were all >0.83. Independent validation of the NNET model in a separate cohort (n=967) produced an AUC of 0.88 (95% CI 0.85 to 0.91) with 74% (95% CI 71 to 77) sensitivity and 92% (95% CI 87 to 96) specificity. CONCLUSIONS: The established NNET TB prediction model discriminated TB from HC, LTBI, RxTB and PN in a large cohort of patients. This diagnostic assay may augment current TB diagnostics.
Assuntos
Ferro/sangue , Tuberculose/diagnóstico , Adolescente , Adulto , Biomarcadores/sangue , Diagnóstico Diferencial , Estudos de Viabilidade , Feminino , Ferritinas/sangue , Homeostase , Humanos , Tuberculose Latente/diagnóstico , Masculino , Pessoa de Meia-Idade , Redes Neurais de Computação , Pneumonia/diagnóstico , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Transferrina/análise , Adulto JovemRESUMO
The sustained increase in the incidence of nontuberculous mycobacterial (NTM) infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification. The MeltPro Myco assay is the first diagnostic system that identifies 19 clinically relevant mycobacteria in a single reaction based on multicolor melting curve analysis run on a real-time PCR platform. The assay was comprehensively evaluated regarding its analytical and clinical performances. The MeltPro Myco assay accurately identified 51 reference mycobacterial strains to the species/genus level and showed no cross-reactivity with 16 nonmycobacterial strains. The limit of detection was 300 bacilli/ml, and 1% of the minor species was detected in the case of mixed infections. Clinical studies using 1,163 isolates collected from five geographically distinct health care units showed that the MeltPro Myco assay correctly identified 1,159 (99.7%) samples. Further testing with 94 smear-positive sputum samples showed that all samples were correctly identified. Additionally, the entire assay can be performed within 3 h. The results of this study confirmed the efficacy of this assay in the reliable identification of mycobacteria, suggesting that it might potentially be used as a screening tool in regions endemic for tuberculosis.
Assuntos
Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Coinfecção/diagnóstico , Coinfecção/microbiologia , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Humanos , Técnicas Microbiológicas/normas , Técnicas de Diagnóstico Molecular/normas , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Escarro/microbiologia , Fatores de Tempo , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
The direct repeat (DR) region in the Mycobacterium tuberculosis (MTB) genome is composed of highly polymorphic direct variant repeats, which are the basis of spacer oligonucleotide typing (spoligotyping) to study the population structure and epidemiology of M. tuberculosis However, the membrane hybridization-based detection format requires various post-PCR manipulations and is prone to carryover contamination, restricting its wide use in high-TB-burden and resource-limited countries. We developed a one-step spoligotyping protocol, termed McSpoligotyping, based on real-time PCR. The typing results can be generated within 3 h by a single step of DNA addition. When evaluated with a collection of 1,968 isolates of MTB, McSpoligotyping agreed 97.71% (1,923/1,968) by sample and 99.93% (84,568/84,624) by spacer with traditional spoligotyping. Sequencing results showed that McSpoligotyping was even more accurate than spoligotyping (99.34% versus 98.37%). Further exploration of the false results of McSpoligotyping revealed the presence of single-nucleotide polymorphisms in the DR region. We concluded that McSpoligotyping could be used in epidemiology studies of tuberculosis by taking advantage of the shortened procedure, ease of use, and compatibility of results with standard spoligotyping.
Assuntos
Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Mycobacterium tuberculosis/classificação , Tuberculose/microbiologia , DNA Bacteriano/genética , Genótipo , Humanos , Tipagem Molecular/normas , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Fatores de TempoRESUMO
Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 105M. tuberculosis genomes/µl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 104M. tuberculosis genomes/µl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Técnicas de Diagnóstico Molecular , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Análise de Sequência de DNA , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
BACKGROUND: Pulmonary nontuberculous mycobacteria (NTM) disease is of increasing public health concern in China. Information is limited regarding risk factors associated with this disease in China. The objective of this study was to describe the epidemiology of pulmonary disease due to NTM in Southern China. METHODS: We retrospectively reviewed the medical records of pulmonary NTM patients registered in the Guangzhou Chest Hospital with positive mycobacterial cultures during 2013-2016. We described sex, age, residence, treatment history, laboratory examination results and comorbidities of pulmonary NTM patients. RESULTS: Among the 607 NTM cases, the most prevalent species were Mycobacterium avium complex (44.5%), Mycobacterium abscessus complex (40.5%), Mycobacterium kansasii (10.0%) and Mycobacterium fortuitum (2.8%). The male:female ratio was significantly lower among patients infected with rapidly growing mycobacteria (RGM) than among those with slowly growing mycobacteria (SGM). The risk of developing SGM disease significantly increased with advancing age. In addition, pulmonary RGM diseases were more common in migrant population than resident population. Notably, patients with pulmonary RGM diseases were significantly more likely to have bronchiectasis underlying noted than those with SGM diseases. No significant difference was observed in in vitro drug susceptibility among NTM species. CONCLUSION: Our data illustrate that the M. avium complex is the most predominant causative agent of pulmonary NTM disease in Southern China. Female, migrant population, the presence of bronchiectasis are independent risk factors for pulmonary diseases due to RGM. In addition, the prevalence of SGM increases significantly with advancing age.
Assuntos
Pneumopatias/epidemiologia , Pneumopatias/microbiologia , Infecções por Mycobacterium não Tuberculosas/complicações , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Antibióticos Antituberculose/uso terapêutico , China/epidemiologia , Comorbidade , Feminino , Humanos , Pneumopatias/tratamento farmacológico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Micobactérias não Tuberculosas , Prevalência , Estudos Retrospectivos , Fatores de Risco , Distribuição por Sexo , Adulto JovemRESUMO
The main goal of our study was to evaluate the in vitro bedaquiline susceptibility of six prevalent species of pathogenic nontuberculous mycobacteria (NTM) in China. In addition, we investigated the potential molecular mechanisms contributing to bedaquiline resistance in the different NTM species. Among slowly growing mycobacteria (SGM), bedaquiline exhibited the highest activity against Mycobacterium avium; the MIC50 and MIC90 values were 0.03 and 16 mg/liter, respectively. Among rapidly growing mycobacteria (RGM), Mycobacterium abscessus subsp. abscessus (M. abscessus) and Mycobacterium abscessus subsp. massiliense (M. massiliense) seemed more susceptible to bedaquiline than Mycobacterium fortuitum, with MIC50 and MIC90 values of 0.13 and >16 mg/liter, respectively, for both species. On the basis of bimodal distributions of bedaquiline MICs, we proposed the following epidemiological cutoff (ECOFF) values: 1.0 mg/liter for SGM and 2.0 mg/liter for RGM. Among M. avium, Mycobacterium intracellulare, Mycobacterium kansasii, M. abscessus, M. massiliense, and M. fortuitum isolates, 14 (29.8%), 41 (27.2%), 33 (39.3%), 44 (20.2%), 42 (25.8%), and 7 (31.8%), respectively, were resistant to bedaquiline. No significant differences in the proportions of bedaquiline resistance among these species were observed (P > 0.05). Genetic mutations were observed in 74 isolates (10.8%), with all nucleotide substitutions being synonymous. In conclusion, our data demonstrate that bedaquiline shows moderate in vitro activity against NTM species. Using the proposed ECOFF values, we could distinguish between bedaquiline-resistant and -susceptible strains with the broth dilution method. In addition, no nonsynonymous mutations in the atpE gene that conferred bedaquiline resistance in all six NTM species were identified.
Assuntos
Antituberculosos/farmacologia , Diarilquinolinas/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/genética , ATPases Bacterianas Próton-Translocadoras/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/análogos & derivados , Cromatografia Líquida de Alta Pressão , DNA de Cadeia Simples/genética , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Pirazinamida/farmacologia , Pirofosfatases/genéticaRESUMO
Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies.
Assuntos
Acetilação , Lisina/metabolismo , Mycobacterium/metabolismo , Processamento de Proteína Pós-Traducional , Aerobiose , Motivos de Aminoácidos , Sítios de Ligação , Metabolismo dos Carboidratos , Cromatografia Líquida , Imunoprecipitação , Espectrometria de Massas em TandemRESUMO
We expressed the wild-type rplC and mutated rplC (Cys154Arg) genes, respectively, in Mycobacterium tuberculosis H37Ra and H37Rv in an attempt to delineate the role of rplC (Cys154Arg) regarding oxazolidinone resistance. An increase of the MICs of linezolid (LZD) and sutezolid (PNU-100480, PNU) against the recombinant mycobacteria with overexpressed rplC mutation (Cys154Arg) was found, suggesting the rplC gene is a determinant of bacillary susceptibilities to LZD and PNU.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/metabolismo , Oxazolidinonas/farmacologia , Proteínas Ribossômicas/metabolismo , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteína Ribossômica L3 , Proteínas Ribossômicas/genéticaRESUMO
Tuberculosis (TB) is one of the three major infectious diseases in China and all over the world. In 2014, for the first time, TB killed more people than HIV did. Non-first line anti-TB drugs are used as main drugs in the treatment of MDR-TB. However, MDR-TB can gradually develop as extensively drug-resistant TB (XDR-TB) because of poor diagnosis, the unreasonable treatment, poor medical conditions and so on. The death rate of XDR-TB is close to lung cancer. Research on the mechanism of drug resistance of Mycobacterium tuberculosis has turned to non first-line anti-TB drugs: second and third line drugs and some new anti-TB drugs in development. In this review, we summarized the drug resistance mechanisms of the common non-first line anti-TB drugs. Most of drug resistant TB patients can't get timely diagnosis and correct treatment. So at the end of this article, we also summarized the common methods to diagnose drug-resistant TB.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3' end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.
Assuntos
Amidoidrolases/genética , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Pirazinamida/farmacologia , Proteínas Ribossômicas/genética , China , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/efeitos dos fármacos , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVE: To study regimens containing isoniazid and rifampicin for the treatment of pulmonary tuberculosis with isoniazid or rifampicin resistance. METHODS: Eighty patients with isoniazid or rifampicin resistance, and whose sputum were still positive at the end of 2-month therapy with isoniazid (H), rifampicin (R), pyrazineamide (Z) and ethambutol (E), were retrospectively analyzed from Jan.2009 to Dec.2012 in Guangzhou Chest Hospital. According to the Mycobacterium drug sensitive test (DST) before the treatment with isoniazid and rifampicin, the patients were divided into the sensitive group (either H or R sensitive), the multidrug-resistance group (both H and R resistance) and the single-resistance group (H or R resistance). There were 80 patients (57 females, 23 males) whose sputum was still positive at the end of 2 month treatment. Their ages ranged from 16-80 (average 45) years. Among them, 29 received the first-treatment, while 51 received retreatment. There were 37 cases in the sensitive group, with 18 first-treatment patients and 19 retreatment patients. There were 15 cases in the single-resistance group, with 3 first-treatment patients and 12 retreatment patients. There were 28 cases in the multidrug-resistance group, with 8 first-treatment patients and 20 retreatment patients. RESULTS: After treatment, mycobacterial conversion to MDR-TB occurred in 2 patients in the sensitive group, and in 6 patients in the single-resistance group. The rate of conversion to MDR-TB was higher in the single-resistance group than that in the sensitive group (χ² = 12.849, P = 0.000).Six patients with single H resistance converted to MDR-TB and 2 patients with single R resistance converted to MDR-TB (P < 0.05, RR = 18.0). CONCLUSIONS: Single H or R drug-resistance was more common in retreated patients with pulmonary tuberculosis.If regimens containing isoniazid and rifampicin was used to treat patients with single H or R drug-resistance, resistant enlarging effect may appear and lead to MDR-TB. The retreated patients should be monitored as soon as possible for detection of Mycobacterium resistance to H and R, and regimens for H or R-resistance should be used to prevent resistant enlarging effect.
Assuntos
Antituberculosos/uso terapêutico , Isoniazida/administração & dosagem , Rifampina/administração & dosagem , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Etambutol , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Retratamento , Estudos Retrospectivos , Escarro , Tórax , Resultado do Tratamento , Adulto JovemRESUMO
Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag+ releasing from C-Ag+-C structure and the fluorescence quenching of CdTe QDs by the released Ag+. Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag+. The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L-1 and 0.33 amol L-1. This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis.