New coumarin linked thiazole derivatives as antimycobacterial agents: Design, synthesis, enoyl acyl carrier protein reductase (InhA) inhibition and molecular modeling.

Tuberculosis is a global serious problem that imposes major health, economic and social challenges worldwide. The search for new antitubercular drugs is extremely important which could be achieved via inhibition of different druggable targets. Mycobacterium tuberculosis enoyl acyl carrier protein reductase (InhA) enzyme is essential for the survival of M. tuberculosis. In this investigation, a series of coumarin based thiazole derivatives was synthesized relying on a molecular hybridization approach and was assessed against thewild typeMtb H37Rv and its mutant strain (ΔkatG) via inhibiting InhA enzyme. Among the synthesized derivatives, compounds 2b, 3i and 3j were the most potent against wild type M. tuberculosis with MIC values ranging from 6 to 8 µg/ mL and displayed low cytotoxicity towards mouse fibroblasts at concentrations 8-13 times higher than the MIC values. The three hybrids could also inhibit the growth of ΔkatGmutant strain which is resistant to isoniazid (INH). Compounds 2b and 3j were able to inhibit the growth of mycobacteria inside human macrophages, indicating their ability to penetrate human professional phagocytes. The two derivatives significantly suppress mycobacterial biofilm formation by 10-15 %. The promising target compounds were also assessed for their inhibitory effect against InhA and showed potent effectiveness with IC50 values of 0.737 and 1.494 µM, respectively. Molecular docking studies revealed that the tested compounds occupied the active site of InhA in contact with the NAD+ molecule. The 4-phenylcoumarin aromatic system showed binding interactions within the hydrophobic pocket of the active site. Furthermore, H-bond formation and π -π stacking interactions were also recorded for the promising derivatives.

The first-in-class pyrazole-based dual InhA-VEGFR inhibitors towards integrated antitubercular host-directed therapy.

Several facets of the host response to tuberculosis have been tapped for clinical investigation, especially targeting angiogenesis mediated by VEGF signaling from infected macrophages. Herein, we rationalized combining the antiangiogenic effects of VEGFR-2 blockade with direct antitubercular InhA inhibition in single hybrid dual inhibitors as advantageous alternatives to the multidrug regimens. Inspired by expanded triclosans, the ether ligation of triclosan was replaced by rationalized linkers to assemble the VEGFR-2 inhibitors thematic scaffold. Accordingly, new series of 3-(p-chlorophenyl)-1-phenylpyrazole derivatives tethered to substituted ureas and their isosteres were synthesized, evaluated against Mycobacterium tuberculosis virulent cell line H37Rv, and assessed for their InhA inhibitory activities. The urea derivatives 8d and 8g exhibited the most promising antitubercular activity (MIC = 6.25 µg/mL) surpassing triclosan (MIC = 20 µg/mL) with potential InhA inhibition, thus identified as the study hits. Interestingly, both compounds inhibited VEGFR-2 at nanomolar IC50 (15.27 and 24.12 nM, respectively). Docking and molecular dynamics simulations presumed that 8d and 8g could bind to their molecular targets InhA and VEGFR-2 posing essential stable interactions shared by the reference inhibitors triclosan and sorafenib. Finally, practical LogP, Lipinski's parameters and in silico ADMET calculations highlighted their drug-likeness as novel leads in the arsenal against TB.

Identification of new anti-mycobacterial agents based on quinoline-isatin hybrids targeting enoyl acyl carrier protein reductase (InhA).

Tuberculosis (TB) is a global issue that poses a significant economic burden as a result of the ongoing emergence of drug-resistant strains. The urgent requirement for the development of novel antitubercular drugs can be addressed by targeting specific enzymes. One such enzyme, Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein (enoyl-ACP) reductase (InhA), plays a crucial role in the survival of the MTB bacterium. In this research study, a series of hybrid compounds combining quinolone and isatin were synthesized and assessed for their effectiveness against MTB, as well as their ability to inhibit the activity of the InhA enzyme in this bacterium. Among the compounds tested, 7a and 5g exhibited the most potent inhibitory activity against MTB, with minimum inhibitory concentration (MIC) values of 55 and 62.5 µg/mL, respectively. These compounds were further evaluated for their inhibitory effects on InhA and demonstrated significant activity compared to the reference drug Isoniazid (INH), with IC50 values of 0.35 ± 0.01 and 1.56 ± 0.06 µM, respectively. Molecular docking studies investigated the interactions between compounds 7a and 5g and the target enzyme, revealing hydrophobic contacts with important amino acid residues in the active site. To further confirm the stability of the complexes formed by 5g and 7a with the target enzyme, molecular dynamic simulations were employed, which demonstrated that both compounds 7a and 5g undergo minor structural changes and remain nearly stable throughout the simulated process, as assessed through RMSD, RMSF, and Rg values.

Exploring the plasticity of the InhA substrate-binding site using new diaryl ether inhibitors.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.

A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

Navigating the Chemical Space of ENR Inhibitors: A Comprehensive Analysis.

Antimicrobial resistance is a global health threat that requires innovative strategies against drug-resistant bacteria. Our study focuses on enoyl-acyl carrier protein reductases (ENRs), in particular FabI, FabK, FabV, and InhA, as potential antimicrobial agents. Despite their promising potential, the lack of clinical approvals for inhibitors such as triclosan and isoniazid underscores the challenges in achieving preclinical success. In our study, we curated and analyzed a dataset of 1412 small molecules recognized as ENR inhibitors, investigating different structural variants. Using advanced cheminformatic tools, we mapped the physicochemical landscape and identified specific structural features as key determinants of bioactivity. Furthermore, we investigated whether the compounds conform to Lipinski rules, PAINS, and Brenk filters, which are crucial for the advancement of compounds in development pipelines. Furthermore, we investigated structural diversity using four different representations: Chemotype diversity, molecular similarity, t-SNE visualization, molecular complexity, and cluster analysis. By using advanced bioinformatics tools such as matched molecular pairs (MMP) analysis, machine learning, and SHAP analysis, we were able to improve our understanding of the activity cliques and the precise effects of the functional groups. In summary, this chemoinformatic investigation has unraveled the FAB inhibitors and provided insights into rational antimicrobial design, seamlessly integrating computation into the discovery of new antimicrobial agents.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone.

OBJECTIVES: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. METHODS: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. RESULTS: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DISCUSSION: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Occurrence of multidrug-resistant Mycobacterium tuberculosis in upper Southern Thailand.

Background and Aim: Mycobacterium tuberculosis causes global concern with tuberculosis (TB). Multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) pose additional challenges, as they resist to multiple first-line drugs. This study investigated the occurrence of TB, antibiotic resistance due to inhA and katG gene mutations, and multidrug resistance in M. tuberculosis during fiscal years 2020-2022. Materials and Methods: Samples were gathered from hospitals in seven provinces of upper Southern Thailand. The study investigated the correlation between inhA and katG gene mutations in M. tuberculosis and the development of antimicrobial resistance and isoniazid resistance. Results: A total of 19,186 samples were sent to the Office of Disease Prevention and Control Region 11st, Nakhon Si Thammarat, Thailand. The results showed that 51% of the samples were obtained from patients located in Nakhon Si Thammarat, followed by Surat Thani provinces. Regarding the spatial distribution of TB-infected cases, the incidence of TB was high in the province, which has a moderate to high population density. The highest average occurrence of TB in this study was found in Phuket province (9.75/100,000 risk person-year). The detected isoniazid resistance was 394, 255, and 179 cases in 2020, 2021, and 2022, respectively. A total of 99 isolates were MDR, whereas four isolates were XDR. The antimicrobial resistance associated with the inhA mutation was 192, 142, and 105 isolates, respectively, whereas the resistance associated with the katG mutation was 249, 182, and 120 cases in 2020, 2021, and 2022, respectively. Conclusion: These findings contribute to the understanding of the occurrence of antibiotic-resistant TB that could lead to use as data for preventing MDR-TB.

Cu-promoted synthesis of triclosan-Mannich and Glaser adducts: anti-mycobacterial evaluation with in silico validations.

Aim: The WHO, Global tuberculosis report 2022 estimated number of tuberculosis (TB) cases reached 10.6 million in 2021, reflecting a 4.5% increase compared with the 10.1 million reported in 2020. The incidence rate of TB showed 3.6% rise from 2020 to 2021. Results/methodology: This manuscript discloses Cu-promoted single pot A3-coupling between triclosan (TCS)-based alkyne, formaldehyde and secondary amines to yield TCS-based Mannich adducts. Additionally, the coupling of TCS-alkynes in the presence of Cu(OAc)2 afforded the corresponding homodimers. Among tested compounds, the most potent one in the series 11 exhibited fourfold higher potency than rifabutin against drug-resistant Mycobacterium abscessus. The selectivity index was also substantially improved, being 26 (day 1) and 15 (day 3), which is four-times better than TCS.

[Box: see text].

Diferencias en los polimorfismos de genes asociados al control del desarrollo folicular entre búfalos (Bubalus bubalis) y vacunos (Bos indicus) / Differences in polymorphisms of genes associated with follicle development control between buffaloes (Bubalus bubalis) and cattle (Bos indicus) / Diferenças nos polimorfismos genéticos associados ao controle do desenvolvimento folicular entre búfalos (Bubalus bubalis) e bovinos (Bos indicus)

Resumen Se han encontrado asociaciones entre polimorfismos genéticos y parámetros reproductivos, este abordaje adicionalmente permitiría proponer explicaciones a diferentes fenómenos estudiados. FSHr, InhA, AMH y AMHr son genes asociados al desarrollo folicular, que tienen una alta homología y la misma función en bovinos. El objetivo del presente trabajo fue evaluar en bovinos muy relacionados si se encuentran algunos polimorfismos asociados a parámetros reproductivos reportados en otras especies. Durante el 2018, en el Magdalena Medio Colombiano fueron tomadas muestras de sangre anticoagulada de 50 búfalas mestizas (Bubalus bubalis) y 50 vacas cebú comercial (Bos indicus). Se registró la paridad, días abiertos, intervalo parto primer servicio y edad al primer parto de cada animal, sin alteraciones anatómicas, con edad y peso similar, mantenidos en las mismas condiciones de alimentación y manejo. Se extrajo el ADN por el método de Salting out y se evaluaron los polimorfismos de acuerdo con lo reportado en la literatura. Se compararon los datos usando la prueba Mann Whitney, se consideró significativo un valor de p<0,05. Se encontró que los búfalos tienen menores niveles de AMH, edad al primer parto, intervalo ente partos que las vacas (p<0,001), mayor paridad (p=0,005). No se confirmaron los po- limorfismos reportados en Holstein y en humanos en ninguna de las muestras y especies analizadas. No confirmar los hallazgos en bovinos de otras razas y espe- cies, aunque las especies tengan diferentes parámetros reproductivos, muestra la necesidad de replantear el abordaje del estudio de la asociación de los fenómenos reproductivos y genéticos.

Abstract Associations have been found between genetic polymorphisms and reproductive parameters, this approach additionally would allow us to propose explanations to the phenomena studied. FSHr, InhA, AMH and AMHr are genes associated with follicular development, which have a high homology and the same function in bovines. The objective of this work was to evaluate if some polymorphisms described in other species associated with reproductive parameters are found in two closely related bovines. During 2018, in the Colombian Magdalena Medio anticoagulated blood samples from 50 buffaloes (Bubalus bubalis) and 50 commercial zebu cows (Bos indicus) were taken. All animals without anatomical abnormalities and similar weight and age were taken and the. parity, intercalving period and age at first calving were also recorded. DNA were extracted using Salting out method and the reported polymorphisms of the above mentioned genes were evaluated. Data were compared using Mann Whitney test and p<0.05 value were considered significant. Buffaloes have lower AMH levels, age at first calving, calving interval than Bos indicus cows (p<0.001) and higher parity (p=0.005). The polymorphisms reported in Holstein and humans were not confirmed in any of the samples and species analyzed. The results, are in some way paradoxical because there are differences in reproductive parameters but nothing different in the studies genes. It shows the need to rethink the approach of the study of the association of reproductive and genetic phenomena

Resumo Foram encontradas associações entre polimorfismos genéticos e parâmetros repro- dutivos; essa abordagem também permitiria propor explicações para os diferentes fenômenos estudados. FSHr, InhA, AMH e AMHr são genes associados ao desenvol- vimento folicular, que apresentam alta homologia e a mesma função em bovinos. O objetivo do presente trabalho foi avaliar em bovinos intimamente relacionados se alguns polimorfismos associados a parâmetros reprodutivos relatados em outras espécies. Em 2018, 50 vacas de búfalo (Bubalus bubalis) e 50 de zebu comerciais (Bos indicus) foram amostradas na Magdalena Medio colombiana. Foram registra- das paridade, dias abertos, intervalo de nascimento do primeiro serviço e idade no primeiro nascimento de cada animal, sem alterações anatômicas, com idade e peso semelhantes, mantidas nas mesmas condições de alimentação e manuseio. O DNA foi extraído pelo método Salting out e os polimorfismos foram avaliados de acordo com o relatado na literatura. Os dados foram comparados pelo teste de Mann Whitney, sendo considerado significativo um valor de p <0,05. Verificou-se que os búfalos apresentam níveis mais baixos de AMH, idade do primeiro nascimento, intervalo entre os nascimentos que as vacas (p <0,001), maior paridade (p = 0,005). Os polimorfis- mos relatados em Holstein e em humanos não foram confirmados em nenhuma das amostras e espécies analisadas. A não confirmação dos achados em bovinos de outras raças e espécies, embora as espécies possuam diferentes parâmetros repro- dutivos, mostra a necessidade de repensar a abordagem do estudo da associação dos fenômenos reprodutivos e genéticos.

Gene mutation associated with drug resistance in M.tuberculosis strains isolated from national TB prevalence survey in Mongolia / Монголын Анагаах Ухаан

Background@#According to the First National Tuberculosis (TB) Prevalence Survey in Mongolia the prevalence of bacteriologically-confirmed pulmonary TB among adults was 559.6 (95% CI: 454.5–664.7) per 100000 population in 2014–2015. This was three times as high as previously estimated. Nationwide anti-tuberculosis (TB) drug resistance survey was conducted in 1999 and 2007 in Mongolia. Share of multidrug resistant TB (MDR-TB) cases among newly notified TB cases increased from 1.0% in 1999 to 1.4% in 2007. Accordingly, we aimed to perform drug susceptibility test on strains isolated from TB Prevalence Survey and to determine the prevalence of drug resistant TB.@*Material and Methods@#All 242 MTB strains isolated from the survey TB cases were tested GenoTypeMTBDRplus test and conventional 1st line DST on solid medium. @*Result@#Conventional DST and GenoTypeMTBDRplus tests done for 93.8% (227/242) of them and 6.2% (15/242) were tested by GenoTypeMTBDRplus only. A 61.6% (95%CI 55.3-67.4) of all cases were susceptible to first line anti-TB drugs, any drug resistance and MDR-TBdetected as 38.4% (95% CI 32.5-44.7)and 9.5% (95% CI 6.4-13.9), respectively. Prevalence of MDR-TB was7.8% (95% CI 4.9-12.4) among new and 17.9% (95% CI 9.0-32.7) among previously treated cases. The 64 strains were identified as a resistant to isoniazid, 32.8% (42/64) and 65.6% (21/64) were katG, and inhAmutation, respectively. One isolate (1.6%) was mutations in both the inhAand katGgenes.The predominant mutations detected in therpoB were S531L (91.3%) among rifampicin resistant isolates and the mutation in inhAwas C–15T (100%) and katG mutation was S315T1 (100%) among isoniazid-resistant isolates. @*Conclusion@#Prevalence of cases with DR-TB is high among prevalent TB cases, especially prevalence of MDR-TB among new cases. In comparison to previous studies, DR-TB cases seem to be increased. Rifampicin resistant strains have a mutation of the rpoBand resistance to isoniazid is predominantly associated with the inhA mutation.

Inhalation properties of the nebulized recombinant human interferon α1b injection / 国际药学研究杂志

Objective: To investigate the aerodynamic characteristics of the nebulized recombinant human inter- feron α1b(rhIFN α1b)injection and its delivery in different respiratory modes both in vitro. Methods: The particle size distribution and aerodynamic properties of the nebulized rhIFN α1b injection for inhalation were evaluated with Spraytec STP5313 and the next generation pharmaceutical impactor(NGI). The total delivered dose and delivery rate were deter- mined using a breathing simulator. Results:After atomization, the D50 of rhIFN α1b droplets was 2.74 μm, the fine par- ticle fraction(FPF)was 77.49%, the mass median aerodynamic diameter(MMAD)was 3.26 μm, and the geometric standard deviation(GSD)was 1.93. In neonatal, infant, and child breathing modes, the delivered total amount of rhIFN α1b by spraying for 220 seconds was 2.10, 2.44, and 3.51 μg, respectively. Conclusion: After atomization, the particle size of rhIFN α1b injection was small enough to be transmitted to the lung, and the total delivered dose and delivery rate showed a tendency of increase in turn in the neonatal, infant, and child breathing modes, indicating that the effective dose of the drug and the age of patients should be considered when formulating the clinical treatment plan.

Correlation between GenoType MTBDRplus Assay and Phenotypic Susceptibility Test for Prothionamide in Patients with Genotypic Isoniazid Resistance / 결핵

BACKGROUND: The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto). METHODS: A total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay. RESULTS: The katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations. CONCLUSION: It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

Insilico Analysis of Phytoconstituents from Allium sativum as Potential Inhibitors of Inha in Mycobacterium tuberculosis

ABSTRACT Tuberculosis is leading cause of death among the global bacterial infections. The main causative for tuberculosis is Mycobacterium tuberculosis, which will survive in its host human being for decades in latent or chronic levels. In addition, the late multidrug resistance at a disturbing rate accompanies the appearance of tuberculosis. The quick spread of resistance to initial stage treatment medications has redirected the focus of the medical community in the creation of an array of new drug against Mycobacterium tuberculosis. The InhA protein is a component of Fatty acid synthetase (FAS) II and exhibits an NADH reliant enoyl-ACP reductase activity. InhA is a vital enzyme of M.tuberculosis in control of cell wall synthesis, which can turn out to be a great focus for the synthesis of anti-tubercular treatment. Inspired from the offering biological actions of phytoconstituents from Allium sativum, the current research concentrates on looking at novel lead compounds from the plant. Molecular docking studies were carried out employing specific phytoconstituents from A.sativum with the protein InhA target. Ajoene shows much more encouragingresults with a Mol Dock rating of 80.6047Kcal/mol, as opposed to the typical initial line drug isoniazid (Moldock score: -58.7028 Kcal/mol). Molecular docking prediction indicate that Ajoene could be formulated into a possible treatment drug for Mycobacterium tuberculosis.

The characteristics of the genes mutations in rifampin and isoniazid resistant Mycobacterium tuberculosis clinical isolates from Baise district, Guangxi autonomous region / 实用医学杂志

Objective To analyze the characteristics of the rpoB, KatG and inhA genes mutations in rifampin and isoniazid resistant Mycobacterium tuberculosis (MTB) clinical isolates in Baise district, Guangxi autonomous region. Methods 128 MTB clinical strains were collected and isolated for drug susceptibility testing, and drug resistant strain DNA was subtracted for rpoB, KatG and inhA genes mutation analysis. Results 75%(27/36)isolates carried mutations in the rpoB gene,and 59.3%(16/27)isolates carried mutations in 531 sites. 44.1%(15/34) isolates carried mutations in KatG or inhA, and 66.7%(10/15) isolates appeared in KatG 315 site, with two new mutations found in KatG 279 and 427 site. In these mutation isolates, 13.3%(2/15) mutations appeared in inhA 5, 6.7%(1/15) in inhA 16, and 20%(3/15) in both katG and inhA. Conclusions The mutation of rpoB, katG and inhA genes in TB is highly correlated with its resistance to rifampin and isoniazid in Baise district, Guangxi autonomous region. The study will provide a basis for further understanding the anti-bacterium mechanism and quick diagnostic methods for drug-resistant tuberculosis.

Curative Effect of Chuankezhi Injection Combined with Budesonide and Formoterol Fumarate Powder for Inhalation in the Treatment of Chronic Obstructive Pulmonary Disease / 中国药师

Objective:To observe the efficacy of Chuankezhi injection combined with budesonide and formoterol fumarate powder for inhalation in the treatment of chronic obstructive pulmonary disease ( COPD) . Methods:Totally 120 patients with COPD were ran-domly divided into two groups:the observation group inhaled budesonide and formoterol fumarate powder for inhalation combined with intramuscular injection of Chuankezhi injection, and the control group inhaled budesonide and formoterol fumarate powder for inhala-tion. The treatment course was 28 days. The pulmonary function indices [ FEV1% expected value, FEV1 /FVC, 6 min walking dis-tance (6MWD)], the times of acute exacerbation and the adverse drug reactions during the treatment were observed and compared be-tween the two groups. Results:After the treatment, the lung function and 6MWD were improved in the two groups, and the improve-ment of FEV1% expected value in the treatment group was more obvious than that in the control group [(59. 7+12. 1)% vs (49. 8+11. 3)%, P<0. 05]. The number of patients with at least once of acute exacerbation was significantly reduced in the treatment group when compared with that in the control group (35% vs 62%, P<0. 01). The incidence of adverse drug reactions in the two groups showed no significant difference. Conclusion:Chuankezhi injection combined with budesonide and formoterol fumarate powder for in-halation can notably improve the lung function in the patients with COPD and reduce the times of acute exacerbation without effect on the incidence of adverse drug reactions.

Detection of Isoniazid and Rifampicin Resistance by Sequencing of katG, inhA, and rpoB Genes in Korea / 대한진단검사의학회지

BACKGROUND: In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods. METHODS: To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INH(R)/RIF(R) (N=30), INH(R)/RIF(S) (N=23), INH(S)/RIF(R) (N=15), and INH(S)/RIF(S) (N=73). RESULTS: Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA -15C>T mutation were susceptible to INH by conventional DST. CONCLUSIONS: Detection of mutations in the katG codon 315, the inhA regulatory region, and the hot-spot region of rpoB would be useful for rapid detection of INH and RIF resistance in Korea.

Mutations of katG and inhA in MDR M. tuberculosis / 결핵및호흡기질환

BACKGROUNDS: Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains. METHODS: PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively. RESULTS: All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 (C-->T) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation (AAA-->AAC) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations. CONCLUSIONS: Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.

Mutations of katG and inhA in MDR M. tuberculosis / 결핵및호흡기질환

BACKGROUNDS: Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains. METHODS: PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively. RESULTS: All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 (C-->T) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation (AAA-->AAC) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations. CONCLUSIONS: Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.