Do Anti-tuberculosis Drugs Reach Their Target?─High-Resolution Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Provides Information on Drug Penetration into Necrotic Granulomas.

Tuberculosis (TB) is characterized by mycobacteria-harboring centrally necrotizing granulomas. The efficacy of anti-TB drugs depends on their ability to reach the bacteria in the center of these lesions. Therefore, we developed a mass spectrometry (MS) imaging workflow to evaluate drug penetration in tissue. We employed a specific mouse model that─in contrast to regular inbred mice─strongly resembles human TB pathology. Mycobacterium tuberculosis was inactivated in lung sections of these mice by γ-irradiation using a protocol that was optimized to be compatible with high spatial resolution MS imaging. Different distributions in necrotic granulomas could be observed for the anti-TB drugs clofazimine, pyrazinamide, and rifampicin at a pixel size of 30 µm. Clofazimine, imaged here for the first time in necrotic granulomas of mice, showed higher intensities in the surrounding tissue than in necrotic granulomas, confirming data observed in TB patients. Using high spatial resolution drug and lipid imaging (5 µm pixel size) in combination with a newly developed data analysis tool, we found that clofazimine does penetrate to some extent into necrotic granulomas and accumulates in the macrophages inside the granulomas. These results demonstrate that our imaging platform improves the predictive power of preclinical animal models. Our workflow is currently being applied in preclinical studies for novel anti-TB drugs within the German Center for Infection Research (DZIF). It can also be extended to other applications in drug development and beyond. In particular, our data analysis approach can be used to investigate diffusion processes by MS imaging in general.

Integrating High-Resolution MALDI Imaging into the Development Pipeline of Anti-Tuberculosis Drugs.

Successful treatment of tuberculosis (TB) requires antibiotics to reach their intended point of action, i.e., necrotizing granulomas in the lung. MALDI mass spectrometry imaging (MSI) is able to visualize the distribution of antibiotics in tissue, but resolving the small histological structures in mice, which are most commonly used in preclinical trials, requires high spatial resolution. We developed a MALDI MSI method to image antibiotics in the mouse lung with high mass resolution (240k @ m/z 200 fwhm) and high spatial resolution (10 µm pixel size). A crucial step was to develop a cryosectioning protocol that retains the distribution of water-soluble drugs in small and fragile murine lung lobes without inflation or embedding. Choice and application of matrices were optimized to detect human-equivalent drug concentrations in tissue, and measurement parameters were optimized to detect multiple drugs in a single tissue section. We succeeded in visualizing the distribution of all current first-line anti-TB drugs (pyrazinamide, rifampicin, ethambutol, isoniazid) and the second-line drugs moxifloxacin and clofazimine. Four of these compounds were imaged for the first time in the mouse lung. Accurate mass identification was confirmed by on-tissue MS/MS. Evaluation of fragmentation pathways revealed the structure of the double-protonated molecular ion of pyrazinamide. Clofazimine was imaged for the first time with 10 µm pixel size revealing clofazimine accumulation in lipid deposits around airways. In summary, we developed a platform to resolve the detailed histology in the murine lung and to reliably detect a range of anti-TB drugs at human-equivalent doses. Our workflow is currently being employed in preclinical mouse studies to evaluate the efficacy of novel anti-TB drugs.

iATP: A Sequence Based Method for Identifying Anti-tubercular Peptides.

BACKGROUND: Tuberculosis is one of the biggest threats to human health. Recent studies have demonstrated that anti-tubercular peptides are promising candidates for the discovery of new anti-tubercular drugs. Since experimental methods are still labor intensive, it is highly desirable to develop automatic computational methods to identify anti-tubercular peptides from the huge amount of natural and synthetic peptides. Hence, accurate and fast computational methods are highly needed. METHODS AND RESULTS: In this study, a support vector machine based method was proposed to identify anti-tubercular peptides, in which the peptides were encoded by using the optimal g-gap dipeptide compositions. Comparative results demonstrated that our method outperforms existing methods on the same benchmark dataset. For the convenience of scientific community, a freely accessible web-server was built, which is available at http://lin-group.cn/server/iATP. CONCLUSION: It is anticipated that the proposed method will become a useful tool for identifying anti-tubercular peptides.

Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, remains the world's deadliest infectious disease. Sterilizing chemotherapy requires at least 6 months of multidrug therapy. Difficulty visualizing the subcellular localization of antibiotics in infected host cells means that it is unclear whether antibiotics penetrate all mycobacteria-containing compartments in the cell. Here, we combined correlated light, electron, and ion microscopy to image the distribution of bedaquiline in infected human macrophages at submicrometer resolution. Bedaquiline accumulated primarily in host cell lipid droplets, but heterogeneously in mycobacteria within a variety of intracellular compartments. Furthermore, lipid droplets did not sequester antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Thus, strong lipid binding facilitated drug trafficking by host organelles to an intracellular target during antimicrobial treatment.

High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types.

Understanding the distribution patterns of antibiotics at the site of infection is paramount to selecting adequate drug regimens and developing new antibiotics. Tuberculosis (TB) lung lesions are made of various immune cell types, some of which harbor persistent forms of the pathogen, Mycobacterium tuberculosis. By combining high resolution MALDI MSI with histology staining and quantitative image analysis in rabbits with active TB, we have mapped the distribution of a fluoroquinolone at high resolution, and identified the immune-pathological factors driving its heterogeneous penetration within TB lesions, in relation to where bacteria reside. We find that macrophage content, distance from lesion border and extent of necrosis drive the uneven fluoroquinolone penetration. Preferential uptake in macrophages and foamy macrophages, where persistent bacilli reside, compared to other immune cells present in TB granulomas, was recapitulated in vitro using primary human cells. A nonlinear modeling approach was developed to help predict the observed drug behavior in TB lesions. This work constitutes a methodological advance for the co-localization of drugs and infectious agents at high spatial resolution in diseased tissues, which can be applied to other diseases with complex immunopathology.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS).

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Development of Inhalable Superparamagnetic Iron Oxide Nanoparticles (SPIONs) in Microparticulate System for Antituberculosis Drug Delivery.

Tuberculosis (TB) is an infectious disease which affects millions of people worldwide. Inhalable polymeric dry powders are promising alternatives as anti-TB drug carriers to the alveoli milieu and infected macrophages, with potential to significantly improve the therapeutics efficiency. Here, the development of a magnetically responsive microparticulate system for pulmonary delivery of an anti-TB drug candidate (P3) is reported. Microparticles (MPs) are developed based on a cast method using calcium carbonate sacrificial templates and incorporate superparamagnetic iron oxide nanoparticles to concentrate MPs in alveoli and enable drug on demand release upon actuation of an external alternate magnetic field (AMF). The MPs are shown to be suitable for P3 delivery to the lower airways and for alveolar macrophage phagocytosis. The developed MPs reveal unique and promising features to be used as an inhalable dry powder allowing the AMF control over dosage and frequency of drug delivery anticipating improved TB treatments.

Visualization of Mycobacterial Biomarkers and Tuberculosis Drugs in Infected Tissue by MALDI-MS Imaging.

MALDI mass-spectrometry imaging (MALDI-MSI) is a technique capable of the label-free identification and visualization of analytes in tissue sections. We have previously applied MALDI-MSI to the study of the spatial distribution of tuberculosis (TB) drugs in necrotic lung granulomas characteristic of pulmonary TB disease, revealing heterogeneous and often suboptimal drug distributions. To investigate the impact of differential drug distributions at sites of infection, we sought to image mycobacterial biomarkers to coregister drugs and bacteria in lesion sections. The traditional method of visualizing Mycobacterium tuberculosis inside lesions is acid-fast staining and microscopy. Directly analyzing and visualizing mycobacteria-specific lipid markers by MALDI-MSI provides detailed molecular information on bacterial distributions within granulomas, complementary to high-spatial-resolution staining and microscopy approaches. Moreover, spatial monitoring of molecular changes occurring in bacteria during granuloma development can potentially contribute to a greater understanding of pulmonary-TB pathogenesis. In this study, we developed a MALDI-MSI method to detect and visualize specific glycolipids of mycobacteria within TB lesions. The biomarker signal correlated well with the bacteria visualized by IHC and acid-fast staining. This observation was seen in samples collected from multiple animal models. Although individual bacteria could not be visualized because of the limit of spatial resolution (50 µm), bacterial clusters were clearly detected and heterogeneously distributed throughout lesions. The ability to visualize drugs, metabolites, and bacterial biomarkers by MALDI-MSI enabled direct colocalization of drugs with specific bacterial target populations (identifiable by distinct metabolic markers). Future applications include assessing drug activity in lesions by visualizing drug-mediated lipid changes and other drug-induced mycobacterial metabolic responses.

Highly sensitive fluorescence biosensors for sparfloxacin detection at nanogram level based on electron transfer mechanism of cadmium telluride quantum dots.

A sensitive fluorescence biosensor for determining sparfloxacin (SPF) based on the electron transfer mechanism and the fluorescence quenching effect of SPF to cadmium telluride quantum dots (CdTe QDs) was developed. The mechanism of the interaction between SPF and CdTe QDs was investigated by UV/Vis absorption and fluorescence spectroscopy. The biosensor could be used for the determination of SPF with a high sensitivity. Under optimum conditions, the linear range was from 0.28 to 40 µg SPF ml(-1) with a correlation coefficient of 0.9983, and the detection limit (3δ/k) was 83.7 ng SPF ml(-1). Furthermore, this method has been applied to the determination of SPF in the synthetic environmental water samples and the spiked human serum samples with good results.

Ion-pair chromatography for simultaneous analysis of ethionamide and pyrazinamide from their porous microparticles.

Ethionamide (ETA) and pyrazinamide (PZA) are considered the drugs of choice for the treatment of multidrug-resistant tuberculosis. Current methods available in the literature for simultaneous determination of ETA and PZA have low sensitivity or involve column modifications with lipophilic cations. The aim of this study was to develop a simple and validated reversed-phase ion-pair HPLC method for simultaneous determination of ETA and PZA for the characterization of polymeric-based porous inhalable microparticles in in vitro and spiked human serum samples. Chromatographic separation was achieved on a Phenomenex C18 column (250 mm × 4.6 mm) using a Shimadzu LC 10 series HPLC. The mobile phase consisted of A: 0.01% trifluoroacetic acid in distilled water and B: ACN/MeOH at 1:1 v/v. Gradient elution was run at a flow rate of 1.5 mL/min and a fixed UV wavelength of 280 nm. The validation characteristics included accuracy, precision, linearity, analytical range, and specificity. Calibration curves at seven levels for ETA and PZA were linear in the analytical range of 0.1-3.0 µg/mL with correlation coefficient of r (2) > 0.999. Accuracy for both ETA and PZA ranged from 94 to 106% at all quality control (QC) standards. The method was precise with relative standard deviation less than 2% at all QC levels. Limits of quantitation for ETA and PZA were 50 and 70 ng/mL, respectively. There was no interference from either the polymeric matrix ions or the biological matrix in the analysis of ETA and PZA.

Determination of isoniazid concentration in rabbit vertebrae by isotope tracing technique in conjunction with HPLC.

Medications compounded with isoniazid (INH) are usually applied to surgical sites at the completion of surgery to locally kill postoperative residual tubercle bacilli. However, the distribution and elimination of INH in the vertebrae in vivo are not known. In this study, isotope tracing was used in conjunction with high-pressure liquid chromatography (HPLC) to address this. INH and technetium-99 m-labeled INH were applied to the vertebrae of rabbits. After 2 and 6 h, osseous tissues containing INH, as determined by radionuclide imaging, were collected for detection with HPLC. The results showed that INH mainly stayed around the vertebrae 6 h after its application and did not permeate widely into the blood or other organs, except for the kidneys. The standard deviations of INH concentrations in the technetium-99 m-INH group were approximately four-fold smaller than those in the INH group. This method of coupling isotope tracing and HPLC can effectively limit experimental error during sample collection, allowing accurate and reliable identification of the concentration levels of INH in osseous tissues in vivo.

Essential oil characterization of two Azorean Cryptomeria japonica populations and their biological evaluations.

Essential oils from foliage, bark and heartwood of Cryptomeriajaponica D. Don from Azores Archipelago (Portugal) were analyzed by GC and GC-MS. Two populations, of black and reddish heartwood color, were studied. The main compounds found in the foliage of both populations were alpha-pinene (9.6-29.5%), (+)-phyllocladene (3.5-26.5%), ent-kaur-16-ene (0.2-20.6%), sabinene (0.5-19.9%) and limonene (1.4-11.5%), with a large variation in individual compounds from each population. Heartwood oils were characterized by a high content of cubebol (2.8-39.9%) and epi-cubebol (4.1-26.9%) isomers, which were absent in the foliage. Elemol and eudesmol isomers were found in the foliage and heartwood oils, while (+)-phyllocladene was absent in heartwood. Black and reddish bark oils were composed of the diterpenes dehydroferruginol (1.9-5.1%) and ferruginol (2.6-11.5%), along with the sesquiterpenes delta-cadinene (10.4-15.9%), alpha-muurolene (3.3-5.4%), epi-zonarene (4.0-5.0%), cubenol (9.3-14.0%), tau-muurolol (4.8-10.7%), beta-eudesmol (3.0-9.9%), gamma-eudesmol (1.9-7.0%) and hedycariol (1.4-6.2%). Azorean C. japonica oils exhibited significant chemical differences compared with native plants from Asia. The essential oils showed moderate antimicrobial activity against the pathogenic fungus Cryptococcus neoformans and human pathogenic bacteria (especially against multidrug-resistant Mycobacterium tuberculosis). The antimicrobial activity of the essential oils may be attributed to compounds such as ent-kaur-16-ene, (+)-phyllocladene, ferruginol and elemol, which are present in different proportions within the complex oil mixture. These results suggest a potential use for C. japonica oils obtained from wood industry leftovers.

Penetration of anti-infective agents into pulmonary epithelial lining fluid: focus on antifungal, antitubercular and miscellaneous anti-infective agents.

Epithelial lining fluid (ELF) is often considered to be the site of extracellular pulmonary infections. During the past 25 years, a limited number of studies have evaluated the intrapulmonary penetration of antifungal, antitubercular, antiparasitic and antiviral agents. For antifungal agents, differences in drug concentrations in ELF or bronchoalveolar lavage (BAL) fluid were observed among various formulations or routes of administration, and between agents within the same class. Aerosolized doses of deoxycholate amphotericin B, liposomal amphotericin B and amphotericin B lipid complex resulted in higher concentrations in ELF or BAL fluid than after intravenous administration. The mean concentrations in ELF following intravenous administration of both anidulafungin and micafungin ranged between 0.04 and 1.38 µg/mL, and the ELF to plasma concentration ratios (based on the area under the concentration-time curve for total drug concentrations) were between 0.18 and 0.22 during the first 3 days of therapy. Among the azole agents, intravenous administration of voriconazole resulted in the highest mean ELF concentrations (range 10.1-48.3 µg/mL) and ratio of penetration (7.1). The range of mean ELF concentrations of itraconazole and posaconazole following oral administration was 0.2-1.9 µg/mL, and the ELF to plasma concentration ratios were <1. A series of studies have evaluated the intrapulmonary penetration of first- and second-line oral antitubercular agents in healthy adult subjects and patients with AIDS. The ELF to plasma concentration ratio was >1 for isoniazid, ethambutol, pyrazinamide and ethionamide. For rifampicin (rifampin) and rifapentine, the ELF to plasma concentration ratio ranged between 0.2 and 0.32, but in alveolar macrophages the concentration of rifampicin was much higher (145-738 µg/mL compared with 3.3-7.5 µg/mL in ELF). No intrapulmonary studies have been conducted for rifabutin. Sex, AIDS status or smoking history had no significant effects on the magnitude of ELF concentrations of antitubercular agents. Subjects who were slow acetylators had higher plasma and ELF concentrations of isoniazid than those who were fast acetylators. Penetration of dapsone into ELF was very good, with the range of mean ELF to plasma concentration ratios being 0.65-2.91 at individual sampling times over 48 hours. Once-daily dosing of aerosolized pentamidine resulted in higher concentrations in BAL fluid than after intravenous administration. The mean BAL concentrations at 15-32 days after once- or twice-monthly administration of aerosolized pentamidine 300 and 600 mg ranged from 6.5 to 28.4 ng/mL. No differences in pentamidine BAL concentrations were observed in symptomatic patients who developed Pneumocystis jirovecii pneumonia compared with patients who did not. Zanamivir concentrations in ELF were similar in magnitude (range 141-326 ng/mL) following administration by continuous intravenous infusion (3 mg/hour), oral inhalation (10 mg every 12 hours) and intravenous bolus (200 mg every 12 hours). Data from case reports have suggested that concentrations of nelfinavir and saquinavir in ELF are undetectable, whereas tipranavir and lopinavir had measureable ELF concentrations (2.20 µmol/L and 14.4 µg/mL, respectively) when these protease inhibitors were co-administrated with ritonavir. While the clinical significance of ELF or BAL concentrations remains unknown for this group of anti-infective agents, the knowledge of drug penetration into the extracellular space of the lung should assist in re-evaluating and designing specific dosing regimens for use against potential pathogens.

Antiprotozoal, antitubercular and cytotoxic potential of cyanobacterial (blue-green algal) extracts from Ireland.

Cyanobacteria (= blue-green algae) are prolific producers of structurally distinct and biologically active metabolites. In the continuation of our search for new sources of anti-infective natural products, we have assessed the in vitro antiprotozoal (Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani) and antitubercular (Mycobacterium tuberculosis) potential of samples of two terrestrial cyanobacteria, Nostoc commune (collected when desiccated and wet) and Rivularia biasolettiana. The cytotoxic potential of the extracts was also evaluated against primary L6 cells. Except for T. cruzi and M. tuberculosis, the crude extracts were active against all the organisms tested and showed no toxicity. The crude extracts were then partitioned between n-hexane, chloroform and aqueous methanol and retested against the same panel of pathogens. The chloroform sub-extracts of both N. commune samples showed significant activity against T. b. rhodesiense (IC50 values 2.0 and 3.5 microg/mL) and P. falciparum (IC50s 7.4 and 5.8 microg/mL), with low toxicity. This trend was also true for R. biasolettiana extracts, and its chloroform sub-extract showed notable activity against all parasitic protozoa. There were differences in the biological activity profiles of extracts derived from desiccated and hydrated forms of N. commune. To our knowledge, this is the first study assessing the anti-infective activity of desiccated and hydrated forms of N. commune, as well as R. biasolettiana. Furthermore, the present work reports such biological activity in terrestrial cyanobacteria from Ireland for the first time. These results warrant the further study of Irish terrestrial cyanobacteria as a valuable source of new natural product leads for the treatment of parasitic protozoal infections.

Composition of three essential oils, and their mammalian cell toxicity and antimycobacterial activity against drug resistant-tuberculosis and nontuberculous mycobacteria strains.

Tuberculosis (TB) is the most ancient epidemic disease in the world and a serious opportunistic disease in HIV/AIDS patients. The increase in multidrug resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) demands the search for novel antimycobacterial drugs. Essential oils (EOs) have been widely used in medicine and some EOs and their major components have been shown to be active against M. tuberculosis. The aim of this work was to evaluate the antimycobacterial and cell toxicity activities of three EOs derived from Salvia aratocensis, Turnera diffusa and Lippia americana, aromatics plants collected in Colombia. The EOs were isolated by hydrodistillation and analyzed by GC/MS techniques. The EOs were tested against 15 Mycobacterium spp using a colorimetric macrodilution method and against mammalian Vero and THP-1 cells by MTT. The activity was expressed as minimal concentration in microg/mL that inhibits growth, and the concentration that is cytotoxic for 50 or 90% of the cells (CC50 and CC90). The major components were epi-alpha-cadinol (20.1%) and 1,10-di-epi-cubenol (14.2%) for Salvia aratocensis; drima-7,9(11)-diene (22.9%) and viridiflorene (6.6%) for Turnera diffusa; and germacrene D (15.4%) and trans-beta- caryophyllene (11.3%) for Lippia americana. The most active EO was obtained from S. aratocensis, with MIC values below 125 microg mL(-1) for M. tuberculosis Beijing genotype strains, and 200 to 500 microg mL(-1) for nontuberculous mycobacterial strains. The EOs were either partially or non toxic to Vero and THP-1 mammalian cells with CC50 values from 30 to > 100 microg mL(-1), and a CC90 > 100 microg mL(-1). The EOs obtained from the three aromatic Colombian plants are an important source of potential compounds against TB. Future studies using the major EO components are recommended.

Analysis and hit filtering of a very large library of compounds screened against Mycobacterium tuberculosis.

There is an urgent need for new drugs against tuberculosis which annually claims 1.7-1.8 million lives. One approach to identify potential leads is to screen in vitro small molecules against Mycobacterium tuberculosis (Mtb). Until recently there was no central repository to collect information on compounds screened. Consequently, it has been difficult to analyze molecular properties of compounds that inhibit the growth of Mtb in vitro. We have collected data from publically available sources on over 300 000 small molecules deposited in the Collaborative Drug Discovery TB Database. A cheminformatics analysis on these compounds indicates that inhibitors of the growth of Mtb have statistically higher mean logP, rule of 5 alerts, while also having lower HBD count, atom count and lower PSA (ChemAxon descriptors), compared to compounds that are classed as inactive. Additionally, Bayesian models for selecting Mtb active compounds were evaluated with over 100 000 compounds and, they demonstrated 10 fold enrichment over random for the top ranked 600 compounds. This represents a promising approach for finding compounds active against Mtb in whole cells screened under the same in vitro conditions. Various sets of Mtb hit molecules were also examined by various filtering rules used widely in the pharmaceutical industry to identify compounds with potentially reactive moieties. We found differences between the number of compounds flagged by these rules in Mtb datasets, malaria hits, FDA approved drugs and antibiotics. Combining these approaches may enable selection of compounds with increased probability of inhibition of whole cell Mtb activity.

Comparative evaluation of the Nitrate Reductase Assay and the Resazurin Microtitre Assay for drug susceptibility testing of Mycobacterium tuberculosis against first line anti-tuberculosis drugs

Tuberculosis remains as a serious infection disease of worldwide distribution, with high morbidity and mortality, mainly in low socio-economic condition countries. The state of emergency of tuberculosis caused by the resistant and multidrug-resistant (MDR) strains, became the main threat to the tuberculosis treatment and control programs. A fast detection method for the resistant strains will allow the implementation of an adequate treatment and contribute for controlling the dissemination of these resistant strains. This study evaluated the performance of the nitrate reductase assay in solid (NRA-LJ) and liquid (NRA-7H9) media, to determine the susceptibility to first line anti-tuberculosis drugs: isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and streptomycin (SMR). Both methods NRA-LJ and NRA-7H9 were evaluated among 18 strains with a known susceptibility profile. The resazurin microtiter assay (REMA) was performed as a reference method. One hundred percent of accordance was observed between NRA-7H9 and REMA for the four tested drugs. When the NRA-LJ method was compared to REMA, the sensitivity and the specificity to INH, RMP, EMB and SMR were 100 percent, 100 percent, 85.7 percent, 76.9 percent and 80 percent, 100 percent, 75 percent and 80 percent, respectively. From the 57 clinical isolates of M. tuberculosis evaluated by NRA-7H9 and REMA, 56 (98.2 percent) were sensitive to all antibiotics tested (INH, RMP, EMB and SMR) by the NRA-7H9 method, while three of these strains were resistant to INH by REMA. One strain showed resistance to INH and RMP for both methods, and MIC of 1.0 µg/ml to INH for both methods, while MIC of 1.0 and 2.0 µg/ml to RMP for REMA and NRA-7H9, respectively. The three assays showed a high level of agreement for rapid detection of rifampicin and isoniazid resistance. Regarding rapidness, the detection of color change in the NRA method is within instants as compared to the overnight incubation required...

A tuberculose permanece como uma séria doença infecciosa, com distribuição mundial, alta morbidade e mortalidade, ocorrendo principalmente em paises com baixa condição econômica. O estado de emergência da tuberculose causada por cepas resistentes e multirresistentes tornou-se uma importante ameaça para o tratamento e programas de controle da tuberculose. Uma rápida detecção de cepas resistentes permitirá a implantação de um tratamento adequado e contribuirá para controlar a disseminação destas cepas. Este estudo avaliou a performace do ensaio nitrato redutase em meio sólido (NRA-LJ) e meio líquido (NRA-7H9), para determinar a sensibilidade frente aos fármacos antituberculosos de primeira linha: isoniazida (INH), rifampicina (RMP), etambutol (EMB) and estreptomicina (SMR). Ambos os métodos, NRA-LJ e NRA-7H9, foram avaliados com 18 cepas com conhecido perfil de sensibilidade. O ensaio de microplaca com resazurina (REMA) foi utilizado como método de referência. A concordância observada entre NRA-7H9 and REMA foi de 100 por cento para os quatro fármacos testados. Quando o método NRA-LJ foi comparado com o REMA, a sensibilidade e especificidade para INH RMP e SMR foram de 100 por cento, 100 por cento, 85,7 por cento, 76,9 por cento e 80 por cento, 100 por cento, 75 por cento and 80 por cento, respectivamente. Dos 57 isolados clinicos de M. tuberculosis avaliados por NRA-7H9 e REMA, 56 (98.2 por cento) foram sensíveis a todos antibióticos testados (INH, RMP, EMB e SMR) pelo método NRA-7H9, enquanto três destas cepas foram resistentes para INH pelo REMA. Uma cepa mostrou resistência para INH e RMP por ambos os métodos, e CMI de 1,0 µg/ml para INH para ambos os métodos, enquanto CMI de 1,0 e 2,0 µg/ml para RMP pelo REMA e NRA-7H9, respectivamente. Os três ensaios mostraram um alto nível ded concordância para uma rápida detecção de resistência a rifampicina e isoniazida. Com relação à rapidez na obtenção dos resultados, a detecção na mudança de...

Intracellular growth and drug susceptibility of Mycobacterium tuberculosis in macrophages.

Intracellular growth of Mycobacterium tuberculosis in human and rabbit monocytes and in mouse and guinea pig macrophages was evaluated. Monocytes or macrophages were infected with M. tuberculosis H37Rv with the multiplicity of infection at 10 mycobacteria per monocyte. The average percentages of infected human and rabbit monocytes were 22% and 19%, while mouse and guinea pig macrophages were 46% and 58%, respectively. The average generation times of M. tuberculosis H37Rv inside human and rabbit monocytes and in mouse and guinea pig macrophages, after culturing the infected cells for 10 days, were 33.4, 50.3, 31.4, and 25.6 h, respectively. Using infected guinea pig macrophages for intracellular evaluation of drug susceptibility, the minimal inhibitory concentrations (MICs) of isoniazid to the intracellular and extracellular M. tuberculosis H37Rv were 0.1 and 0.4 microg/ml, while the MICs of rifampicin were 0.1 and 0.2 microg/ml, respectively. The minimal bactericidal concentrations (MBCs) of isoniazid to the intracellular and extracellular H37Rv were 0.2 and 0.4 microg/ml, while the MCSs of rifampicin were 0.1 and 0.2 microg/ml, respectively.