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1.
Mikrochim Acta ; 191(8): 456, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38980419

RESUMO

Polydopamine (PDA) has garnered significant interest for applications in biosensors, drug delivery, and tissue engineering. However, similar polycatecholamines like polynorepinephrine (PNE) with additional hydroxyl groups and poly-α-methylnorepinephrine (PAMN) with additional hydroxyl and methyl groups remain unexplored in the biosensing domain. This research introduces three innovative biosensing platforms composed of ternary nanocomposite based on reduced graphene oxide (RGO), gold nanoparticles (Au NPs), and three sister polycatecholamine compounds (PDA, PNE, and PAMN). The study compares and evaluates the performance of the three biosensing systems for the ultrasensitive detection of Mycobacterium tuberculosis (MTB). The formation of the nanocomposites was meticulously examined through UV-Visible, Raman, XRD, and FT-IR studies with FE-SEM and HR-TEM analysis. Cyclic voltammetry and differential pulse voltammetry measurements were also performed to determine the electrochemical characteristics of the modified electrodes. Electrochemical biosensing experiments reveal that the RGO-PDA-Au, RGO-PNE-Au, and RGO-PAMN-Au-based biosensors detected target DNA up to a broad detection range of 0.1 × 10-8 to 0.1 × 10-18 M, with a low detection limit (LOD) of 0.1 × 10-18, 0.1 × 10-16, and 0.1 × 10-17 M, respectively. The bioelectrodes were proved to be highly selective with excellent sensitivities of 3.62 × 10-4 mA M-1 (PDA), 7.08 × 10-4 mA M-1 (PNE), and 6.03 × 10-4 mA M-1 (PAMN). This study pioneers the exploration of two novel mussel-inspired polycatecholamines in biosensors, opening avenues for functional nanocoatings that could drive further advancements in this field.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Ouro , Grafite , Indóis , Limite de Detecção , Nanopartículas Metálicas , Polímeros , Técnicas Biossensoriais/métodos , Indóis/química , Polímeros/química , Técnicas Eletroquímicas/métodos , Grafite/química , Ouro/química , Animais , Nanopartículas Metálicas/química , Mycobacterium tuberculosis , Bivalves/química , Nanocompostos/química , Eletrodos , Norepinefrina/análise
2.
Nat Commun ; 15(1): 5740, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38982040

RESUMO

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.


Assuntos
Proteínas de Bactérias , Glicosídeo Hidrolases , Lipopolissacarídeos , Macrófagos , Mananas , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Lipopolissacarídeos/metabolismo , Mananas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicosídeo Hidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Animais , Camundongos , Humanos , Fosfatidilinositóis/metabolismo , Cápsulas Bacterianas/metabolismo
3.
BMC Infect Dis ; 24(1): 684, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38982340

RESUMO

INTRODUCTION: Tuberculous lymphadenitis (TBLN) is an infection of the lymph node caused by Mycobacterium tuberculosis. Histological diagnoses of presumptive patients are often accompanied by cytomorphological features. However, the sensitivities of these features are often precluded by the variable degrees of narrative similarities compared to other diagnostic modalities. OBJECTIVE: The aim of this study was to investigate and compare the cytomorphological and clinical features of presumptive TBLN patients with bacteriological detection methods. METHODS: A similar cohort of TBLN patients from our previous study who were enrolled prospectively from the ALERT Specialized Hospital, Addis Ababa, Ethiopia, was considered for this analysis. SPSS version 26 was used for data analysis. Descriptive analysis was conducted to characterize the study population using the independent variable and presented with frequency tables. The chi-square test was used to measure the association. A P-value of < 0.05 was considered statistically significant. RESULTS: Using FNAC, 60/126 (47.6%) of the participants were reported to have features consistent with TB. Of the total FNAC-positive cases, many (30/60 and 27/60) showed pattern B (caseous necrosis only) and pattern C (epithelioid granuloma with caseous necrosis), respectively. Strong concordance was observed in Pattern A (abundant caseous necrosis with few epithelioid macrophages) followed by patterns B and C with GeneXpert and MGIT culture (P value < 0.001). Night sweats and alcohol intake were shown to correlate with positive cases as reported by FNAC (P value = 0.008 respectively), GeneXpert (P value = 0.02 & 0.001), and culture methods (P-value = < 0.001 & 0.002). CONCLUSION: Cytomorphological features, particularly patterns A, B, and C, could be considered in the diagnosis of TBLN given their comparable outcomes with bacteriological detection methods. On another note, we recommend that due care and attention be given when treating TBLN patients based solely on clinical presentation, as these diagnostics may be prone to false results, leading to inappropriate administration of anti-TB drugs and other consequences.


Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Humanos , Tuberculose dos Linfonodos/diagnóstico , Tuberculose dos Linfonodos/microbiologia , Tuberculose dos Linfonodos/patologia , Masculino , Feminino , Adulto , Estudos Transversais , Mycobacterium tuberculosis/isolamento & purificação , Adulto Jovem , Etiópia , Adolescente , Pessoa de Meia-Idade , Linfonodos/patologia , Linfonodos/microbiologia , Biópsia por Agulha Fina , Criança , Estudos Prospectivos , Idoso , Técnicas Bacteriológicas/métodos
4.
J Med Microbiol ; 73(7)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38973691

RESUMO

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.


Assuntos
Antituberculosos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Nebramicina , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Humanos , Antituberculosos/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Farmacorresistência Bacteriana Múltipla
5.
Front Immunol ; 15: 1321657, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38975346

RESUMO

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.


Assuntos
Macrófagos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Vacinas contra a Tuberculose , Tuberculose , Animais , Mycobacterium tuberculosis/imunologia , Camundongos , Macrófagos/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Inflamassomos/imunologia , Feminino , Vacina BCG/imunologia , Autofagia/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Modelos Animais de Doenças
6.
Med Microbiol Immunol ; 213(1): 14, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38977511

RESUMO

Mycobacterium tuberculosis, a lethal pathogen in human history, causes millions of deaths annually, which demands the development of new concepts of drugs. Considering this fact, earlier research has explored the anti-tuberculosis potential of a probiotic strain, Lactocaseibacillus rhamnosus PMC203, leading to a subsequent focus on the molecular mechanism involved in its effect, particularly on autophagy. In this current study, immunoblotting-based assay exhibited a remarkable expression of autophagy marker LC3-II in the PMC203 treated group compared to an untreated group. A remarkable degradation of p62 was also noticed within treated cells compared to control. Furthermore, the immunofluorescence-based assay showed significant fold change in fluorescence intensity for alexa-647-LC3 and alexa-488-LC3, whereas p62 was degraded noticeably. Moreover, lysosomal biogenesis generation was elevated significantly in terms of LAMP1 and acidic vesicular organelles. As a result, PMC203-induced autophagy played a vital role in reducing M. tuberculosis burden within the macrophages in treated groups compared to untreated group. A colony -forming unit assay also revealed a significant reduction in M. tuberculosis in the treated cells over time. Additionally, the candidate strain significantly upregulated the expression of autophagy induction and lysosomal biogenesis genes. Together, these results could enrich our current knowledge of probiotics-mediated autophagy in tuberculosis and suggest its implications for innovatively managing tuberculosis.


Assuntos
Autofagia , Lacticaseibacillus rhamnosus , Macrófagos , Mycobacterium tuberculosis , Probióticos , Mycobacterium tuberculosis/genética , Lacticaseibacillus rhamnosus/fisiologia , Lacticaseibacillus rhamnosus/metabolismo , Macrófagos/microbiologia , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Carga Bacteriana , Tuberculose/microbiologia
7.
Nat Commun ; 15(1): 5710, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977711

RESUMO

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Pulmão , Macrófagos Alveolares , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/fisiologia , Animais , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD4-Positivos/imunologia , Camundongos , Feminino , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Macrófagos/imunologia , Macrófagos/microbiologia , Humanos
8.
Sci Rep ; 14(1): 15680, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977729

RESUMO

Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB and MTB-Rif Dx (TN), for detection of Mycobacterium tuberculosis and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, Overall sensitivity of GX was 67.6%, while that of TN was 62.3%. Highest sensitivity by TN was observed in pus samples (89%) and highest specificity (92%) in CSF samples, similar to GX. TN sensitivity was better in fluid and biopsy samples and slightly inferior for lymph node aspirates compared to GX. TN sensitivity for RIF resistance detection was slightly superior to GX. TN and GX results were further compared to Clinical Reference Standards. TN detected 170 TB patients initiated on treatment missed by GX, while GX detected 113 such patients missed by TN. Of 124 samples with RIF resistance discordance between GX and TN, GX reported 103/124 as sensitive, 3/124 as indeterminate and 18 as resistant (13/18 samples had low/very low DNA load) while TN reported RIF resistance indeterminate in 103/111 low/very low DNA load samples. Due to paucibacillary nature of EPTB samples, culture yield was poor and phenotypic drug susceptibility testing failed to resolve the discordance. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.


Assuntos
Mycobacterium tuberculosis , Rifampina , Sensibilidade e Especificidade , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Rifampina/uso terapêutico , Adulto , Feminino , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Idoso , Adulto Jovem , Tuberculose Extrapulmonar
9.
J Med Case Rep ; 18(1): 324, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38978087

RESUMO

BACKGROUND: Mycobacterium tuberculosis is the second most common infectious cause of death in adults worldwide. The ability of this organism to efficiently establish latent infection has enabled it to spread to nearly one-third of individuals worldwide. Approximately 8 million new cases of active tuberculosis disease occur each year, leading to about 1.7 million deaths. The disease incidence is magnified by the concurrent epidemic of human immunodeficiency virus infection. A total of 1.3 million people died from tuberculosis in 2022. In 2022, an estimated 10.6 million people fell ill with tuberculosis worldwide, including 5.8 million men, 3.5 million women, and 1.3 million children. We report a case of thyroid tuberculosis presenting as multinodular goiter. Neck ultrasound was done and revealed abscess collection on the background of multinodular colloid goiter. The diagnosis of thyroid tuberculosis was confirmed by a positive GeneXpert of the pus sample and the presence of extensive caseous necrosis on cytopathology examination. Furthermore, anterior neck swelling may provide a diagnostic challenge by clinically mimicking multinodular goiter or thyroid neoplasms. Owing to its rarity and its tendency to pose a clinical diagnostic challenge, we decided to report it. CASE PRESENTATION: A 60-year-old retired female Ethiopian high-school teacher presented to University of Gondar Hospital, Gondar, Ethiopia with firm, nontender multinodular anterior neck swelling measuring at largest 2 × 3 cm that moves with swallowing. GeneXpert of the pus sample and cytopathology examination confirmed the diagnosis of thyroid tuberculosis, and the patient was started on 2 rifampicin-ethambutol-isoniazid-pyrazinamide/4 rifampicin-isoniazid 3 tablets by mouth/day, which is defined as the preferred first-line anti-tuberculosis regimen in Ethiopia, and pyridoxine 50 mg by mouth per day for 6 months. Since then, she has been followed with regular liver function tests. The patient has shown a smooth course with no significant adverse effects encountered. Currently, the patient has completed her anti-tuberculosis treatment and is doing well. CONCLUSION: In the clinical evaluation of a patient with anterior neck swelling, tuberculosis must be considered as a differential diagnosis in subjects from endemic areas for early diagnostic workup and management.


Assuntos
Antituberculosos , Bócio Nodular , Humanos , Feminino , Diagnóstico Diferencial , Pessoa de Meia-Idade , Bócio Nodular/diagnóstico , Antituberculosos/uso terapêutico , Tuberculose Endócrina/diagnóstico , Tuberculose Endócrina/tratamento farmacológico , Ultrassonografia , Mycobacterium tuberculosis/isolamento & purificação
10.
J Med Primatol ; 53(4): e12722, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38949157

RESUMO

BACKGROUND: Tuberculosis (TB) kills approximately 1.6 million people yearly despite the fact anti-TB drugs are generally curative. Therefore, TB-case detection and monitoring of therapy, need a comprehensive approach. Automated radiological analysis, combined with clinical, microbiological, and immunological data, by machine learning (ML), can help achieve it. METHODS: Six rhesus macaques were experimentally inoculated with pathogenic Mycobacterium tuberculosis in the lung. Data, including Computed Tomography (CT), were collected at 0, 2, 4, 8, 12, 16, and 20 weeks. RESULTS: Our ML-based CT analysis (TB-Net) efficiently and accurately analyzed disease progression, performing better than standard deep learning model (LLM OpenAI's CLIP Vi4). TB-Net based results were more consistent than, and confirmed independently by, blinded manual disease scoring by two radiologists and exhibited strong correlations with blood biomarkers, TB-lesion volumes, and disease-signs during disease pathogenesis. CONCLUSION: The proposed approach is valuable in early disease detection, monitoring efficacy of therapy, and clinical decision making.


Assuntos
Biomarcadores , Aprendizado Profundo , Macaca mulatta , Mycobacterium tuberculosis , Tomografia Computadorizada por Raios X , Animais , Biomarcadores/sangue , Tomografia Computadorizada por Raios X/veterinária , Tuberculose/veterinária , Tuberculose/diagnóstico por imagem , Modelos Animais de Doenças , Tuberculose Pulmonar/diagnóstico por imagem , Masculino , Feminino , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/microbiologia , Doenças dos Macacos/diagnóstico por imagem , Doenças dos Macacos/microbiologia
11.
Methods Mol Biol ; 2833: 23-33, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949697

RESUMO

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.


Assuntos
Descoberta de Drogas , Inativação Gênica , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Descoberta de Drogas/métodos , Humanos , Sistemas CRISPR-Cas , Antituberculosos/farmacologia , Antibacterianos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Farmacorresistência Bacteriana/genética , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico
12.
Methods Mol Biol ; 2833: 65-77, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949702

RESUMO

Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug combinations. Potentiation of PZA could be used to reduce the emergence of resistance, shorten treatment times, and lead to a reduction in the quantity of PZA consumed by patients, thereby reducing the toxic effects. Acidified medium is required for the activity of PZA against Mycobacterium tuberculosis. In vitro assessments of pyrazinamide activity are often avoided because of the lack of standardization, which has led to a lack of effective in vitro tools for assessing and/or enhancing PZA activity.We have developed and optimized a novel, robust, and reproducible, microtiter plate assay, that centers around acidity levels that are low enough for PZA activity. The assay can be applied to the evaluation of novel compounds for the identification of potentiators that enhance PZA activity. In this assay, potentiation of PZA is demonstrated to be statistically significant with the addition of rifampicin (RIF), which can, therefore, be used as a positive control. Conversely, norfloxacin demonstrates no potentiating activity with PZA and can be used as a negative control. The method, and the associated considerations, described here, can be adapted in the search for potentiators of other antimicrobials.


Assuntos
Antituberculosos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Pirazinamida , Pirazinamida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Sinergismo Farmacológico , Rifampina/farmacologia , Humanos
13.
Methods Mol Biol ; 2833: 121-128, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949706

RESUMO

Going back in time through a phylogenetic tree makes it possible to evaluate ancestral genomes and assess their potential to acquire key polymorphisms of interest over evolutionary time. Knowledge of this kind may allow for the emergence of key traits to be predicted and pre-empted from currently circulating strains in the future. Here, we present a novel genome-wide survival analysis and use the emergence of drug resistance in Mycobacterium tuberculosis as an example to demonstrate the potential and utility of the technique.


Assuntos
Mycobacterium tuberculosis , Filogenia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Genoma Bacteriano , Humanos , Evolução Molecular , Farmacorresistência Bacteriana/genética , Tuberculose/microbiologia , Tuberculose/genética
14.
Methods Mol Biol ; 2833: 145-152, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949708

RESUMO

Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries.


Assuntos
Contenção de Riscos Biológicos , Mycobacterium tuberculosis , Escarro , Tiocianatos , Mycobacterium tuberculosis/isolamento & purificação , Humanos , Contenção de Riscos Biológicos/métodos , Escarro/microbiologia , Carga Bacteriana/métodos , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Guanidinas , Temperatura Alta , Viabilidade Microbiana
15.
Methods Mol Biol ; 2833: 153-160, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949709

RESUMO

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.


Assuntos
Carga Bacteriana , Fezes , Mycobacterium tuberculosis , Tuberculose , Fezes/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/genética , Humanos , Carga Bacteriana/métodos , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico , Antituberculosos/uso terapêutico , Antituberculosos/farmacologia , DNA Bacteriano/genética , Escarro/microbiologia
16.
Methods Mol Biol ; 2833: 195-210, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949712

RESUMO

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.


Assuntos
Antituberculosos , Biologia Computacional , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biologia Computacional/métodos , Genoma Bacteriano , Farmacorresistência Bacteriana/genética , Mutação , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico
17.
Methods Mol Biol ; 2833: 185-193, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949711

RESUMO

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.


Assuntos
Genoma Bacteriano , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biblioteca Gênica , DNA Bacteriano/genética , Tuberculose/microbiologia , Tuberculose/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos
18.
Front Immunol ; 15: 1422836, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38947330

RESUMO

Introduction: Neutrophils play a complex and important role in the immunopathology of TB. Data suggest they are protective during early infection but become a main driver of immunopathology if infection progresses to active disease. Neutrophils are now recognized to exist in functionally diverse states, but little work has been done on how neutrophil states or subsets are skewed in TB disease. Methods: To address this, we carried out comprehensive phenotyping by flow cytometry of neutrophils in the blood and airways of individuals with active pulmonary TB with and without HIV co-infection recruited in Durban, South Africa. Results: Active TB was associated with a profound skewing of neutrophils in the blood toward phenotypes associated with activation and apoptosis, reduced phagocytosis, reverse transmigration, and immune regulation. This skewing was also apparently in airway neutrophils, particularly the regulatory subsets expressing PDL-1 and LOX-1. HIV co-infection did not impact neutrophil subsets in the blood but was associated with a phenotypic change in the airways and a reduction in key neutrophil functional proteins cathelicidin and arginase 1. Discussion: Active TB is associated with profound skewing of blood and airway neutrophils and suggests multiple mechanisms by which neutrophils may exacerbate the immunopathology of TB. These data indicate potential avenues for reducing neutrophil-mediated lung pathology at the point of diagnosis.


Assuntos
Infecções por HIV , Imunofenotipagem , Neutrófilos , Tuberculose Pulmonar , Humanos , Neutrófilos/imunologia , Masculino , Adulto , Feminino , Infecções por HIV/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , África do Sul , Coinfecção/imunologia , Pessoa de Meia-Idade , Fenótipo , Citometria de Fluxo , Adulto Jovem , Mycobacterium tuberculosis/imunologia
19.
Front Public Health ; 12: 1244353, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38947352

RESUMO

Introduction: The Eastern Mediterranean Regional Office (EMRO) region accounts for almost 8% of all global Mycobacterium tuberculosis (TB) cases, with TB incidence rates ranging from 1 per 100,000 per year in the United Arab Emirates (UAE) to 204 per 100,000 in Djibouti. The national surveillance data from the Middle East and North Africa (MENA) region on the epidemiology and antimicrobial resistance trends of TB, including MDR-TB remains scarce. Methods: A retrospective 12-year analysis of N = 8,086 non-duplicate diagnostic Mycobacterium tuberculosis complex (MTB complex) isolates from the UAE was conducted. Data were generated through routine patient care during the 2010-2021 years, collected by trained personnel and reported by participating surveillance sites to the UAE National Antimicrobial Resistance (AMR) Surveillance program. Data analysis was conducted with WHONET, a windows-based microbiology laboratory database management software developed by the World Health Organization Collaborating Center for Surveillance of Antimicrobial Resistance, Boston, United States (https://whonet.org/). Results: A total of 8,086 MTB-complex isolates were analyzed. MTB-complex was primarily isolated from respiratory samples (sputum 80.1%, broncho-alveolar lavage 4.6%, pleural fluid 4.1%). Inpatients accounted for 63.2%, including 1.3% from ICU. Nationality was known for 84.3% of patients, including 3.8% Emiratis. Of UAE non-nationals, 80.5% were from 110 countries, most of which were Asian countries. India accounted for 20.8%, Pakistan 13.6%, Philippines 12.7%, and Bangladesh 7.8%. Rifampicin-resistant MTB-complex isolates (RR-TB) were found in 2.8% of the isolates, resistance to isoniazid, streptomycin, pyrazinamide, and ethambutol, was 8.9, 6.9, 3.4 and 0.4%, respectively. A slightly increasing trend of resistance among MTB-complex was observed for rifampicin from 2.5% (2010) to 2.8% (2021). Conclusion: Infections due to MTB-complex are relatively uncommon in the United Arab Emirates compared to other countries in the MENA region. Most TB patients in the UAE are of Asian origin, mainly from countries with a high prevalence of TB. Resistance to first line anti-tuberculous drugs is generally low, however increasing trends for MDR-TB mainly rifampicin linked resistance is a major concern. MDR-TB was not associated with a higher mortality, admission to ICU, or increased length of hospitalization as compared to non-MDR-TB.


Assuntos
Tuberculose Resistente a Múltiplos Medicamentos , Emirados Árabes Unidos/epidemiologia , Humanos , Estudos Retrospectivos , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Masculino , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Feminino , Adulto , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Pessoa de Meia-Idade , Farmacorresistência Bacteriana , Adolescente , Testes de Sensibilidade Microbiana , Adulto Jovem , Vigilância da População
20.
Front Cell Infect Microbiol ; 14: 1410015, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38957797

RESUMO

Background: Tuberculosis (TB) persists as a global health challenge, with its treatment hampered by the side effects of long-term combination drug therapies and the growing issue of drug resistance. Therefore, the development of novel therapeutic strategies is critical. This study focuses on the role of immune checkpoint molecules (ICs) and functions of CD8+ T cells in the search for new potential targets against TB. Methods: We conducted differential expression genes analysis and CD8+ T cell functional gene analysis on 92 TB samples and 61 healthy individual (HI) samples from TB database GSE83456, which contains data on 34,603 genes. The GSE54992 dataset was used to validated the findings. Additionally, a cluster analysis on single-cell data from primates infected with mycobacterium tuberculosis and those vaccinated with BCG was performed. Results: The overexpression of LAG-3 gene was found as a potentially important characteristic of both pulmonary TB (PTB) and extrapulmonary TB (EPTB). Further correlation analysis showed that LAG-3 gene was correlated with GZMB, perforin, IL-2 and IL-12. A significant temporal and spatial variation in LAG-3 expression was observed in T cells and macrophages during TB infection and after BCG vaccination. Conclusion: LAG-3 was overexpressed in TB samples. Targeting LAG-3 may represent a potential therapeutic target for tuberculosis.


Assuntos
Antígenos CD , Linfócitos T CD8-Positivos , Proteína do Gene 3 de Ativação de Linfócitos , Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/genética , Linfócitos T CD8-Positivos/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia , Animais , Antígenos CD/genética , Vacina BCG/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Interleucina-2/metabolismo , Interleucina-2/genética , Perfilação da Expressão Gênica , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Interleucina-12/genética , Interleucina-12/metabolismo , Perforina/genética , Perforina/metabolismo , Masculino
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