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1.
The activity of a calmodulin antagonist, trifluoperazine (TFP), was testedin vitro againstMycobacterium avium (ATCC 25291). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of this compound forM. avium were 20 and 30 μg/ml, respectively. TFP was also found to completely inhibit the growth of 10 isolates ofM. avium (5 patient isolates and 5 environmental isolates) at 30 μg/ml. At near neutral pH (6.8), the MIC of TFP was found to be 20 μg/ml. However, at pH 5.5 (intracellular pH of macrophages), there was a decrease in the inhibitory activity of the compound against this organism. Interestingly, 99.6% ofM. avium within human monocyte-derived macrophages were killed at a drug concentration of 30 μg/ml, which correlates well with the MBC of TFP againstM. avium in vitro. Although the MIC for TFP appears to be higher than that forMycobacterium tuberculosis H37Rv, our studies suggest that calmodulin antagonists might be useful as drugs against infection due toM. avium. It is suggested that administration of TFP in combination with other known drugs may enhance the overall bactericidal effect.  相似文献   

2.
The antibody response to the 38kDa, 16kDa and Lipoarabinomannan (LAM) antigens ofMycobacterium tuberculosis was evaluated using three different ELISAs based on these antigens. The study group included tuberculosis patients (n=52), patients with HIV and TB co-infection (n=10), other chest symptomatics (n=5), HIV infected individuals (n=10), leprosy cases (n=7) and healthy controls (n=75). The results indicate that the 38kDa and LAM based ELISA for IgM/IgG has a low specificity (ranging from 69–85%) and sensitivity (ranging from 55–78%). When three ELISAs are carried out on a single patient the probability of detection of tuberculosis was significantly increased to 95.2% indicating that a single ELISA test is of low sensitivity and that a combination of ELISA’s may be needed to be of any value as a diagnostic test for tuberculosis. Additionally, a western blot assay of the serum antibody response to protein fraction ofM. tuberculosis was analysed in 15 tuberculosis patients and five healthy controls. A multiple antibody response to various M.tuberculosis proteins was observed which varied from patient to patient as compared to controls who showed a single 38–39 kDa protein band positivity. These finding suggest that a western blot assay which determines the antibody response to a set of antigenic components ofM. tuberculosis could be a better serological test for the diagnosis of tuberculosis in our population.  相似文献   

3.
Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.  相似文献   

4.
There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H37Ra and H37Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H37Ra and H37Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM.  相似文献   

5.
We have developed a simple, economical and reproducible method for processing blood samples from HIV infected patients for diagnosis of tuberculosis. The procedure was validated on 55 samples selected for tuberculosis based on clinical criteria. 52 patients had radiological changes indicative of pulmonary tuberculosis of which only 28 were positive for AFB in sputum (sensitivity 54%) and 27 for tuberculin (sensitivity 52%). 26 HIV positive patients who showed positive X-ray did not react to tuberculin. The genus PCR probe missed 3 samples (sensitivity 94%) compared to X-ray.M.tuberculosis was detected in the blood of all X-ray positive cases by PCR using TB400 probe (sensitivity 100%) and another probe forM. tuberculosis, IS6110, missed 6 of them (sensitivity 88% compared to X-ray and 89% compared to TB400). It is proposed that this simple sample processing method could be used to screen all blood samples quickly for mycobacteremia using the genus PCR and only those positive for mycobacteria need to be tested forM.tuberculosis. This would save the scarce resources and time by reducing significantly the number of samples to be screened for species confirmation.  相似文献   

6.
The effect of the antitubercular drugs isoniazid (10 μg/ml), ethambutol (10 μg/ml), rifampicin (0.5 μg/ml) and streptomycin (1 μg/ml) on the calmodulin like protein (CAMLP) content ofMycobacterium tuberculosis H37Rv andM. tuberculosis H37Ra was investigated. The drugs were added to actively growing cells at their mid log phase of growth (14 days) and after 12 more hours of incubation, CAMLP was estimated. In both the mycobacteria, all the four antitubercular drugs CAMLP.  相似文献   

7.
Genomic DNA from a clinical isolate ofMycobacterium avium-intracellulare complex was purified and cloned in PBR 322 at the tetracycline resistance site using Bam HI restriction enzyme. A 16 kb cloned fragment was purified, radiolabeled and used as a probe. Genomic DNA isolated from nineteen MAC strains, threeM. tuberculosis strains and oneM. kansasii strain were digested with Eco RI restriction enzyme, Southern blotted and hybridized with the 16 kb cloned and labeled fragment. Twelve MAC strains showed positive hybridization although five strains gave faint signals. Positive hybridization was noted in two out of the threeM. tuberculosis strains, possibly due to shared DNA homology. No signal was received from the singleM. kansasii strain used in this study.  相似文献   

8.
Tuberculosis remains a major public health problem globally, with India being one of the high burden countries. The common causative agent is Mycobacterium tuberculosis but in developing countries M. bovis is reported as a potential human pathogen. Almost 20% of all reported cases of tuberculosis are of extra pulmonary form of disease. Diagnosis of extra pulmonary tuberculosis (EPTB) is not always possible with conventional methods, due to the long time required and the paucibacillary nature of samples; hence the need of rapid molecular methods. A prospective study was conducted on 300 patients of EPTB over a period of 5 years. These patients were suspected cases of tubercular meningitis, tubercular ascites and tubercular lymphadenitis. Samples analyzed were cerebrospinal fluid, ascitic fluid and lymph node fine needle aspirate. A two step PCR targeting hup B gene was used. Clinical response to anti tubercular therapy (ATT) was taken as positive (gold standard). PCR for hup B gene was positive in 147 samples out of 155 ATT responders. Of these 85.71% were infected with M. tuberculosis, 9.52% with M. bovis alone and 4.76% showed co infection with both M.tb and M. bovis. The sensitivity and specificity of PCR was 90.32 and 94.48% respectively.  相似文献   

9.
Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H37Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.  相似文献   

10.
R-plasmids that transfer antibiotic resistance are common in the non-pathogenicEscherichia coli of the gastro-intestinal tract of human beings and domestic animals, which inturn may enter into sewage. Therefore we have isolated 30Escherichia coli isolates from hospital sewage of Aligarh city. These isolates were tested for their resistance and sensitivity against 10 antibiotics. 90% isolates showed resistance against ampicillin and sulphamethizole. Of the total 30E. coli isolates 86.6% were resistant to erythromycin and rifampicin but none of them was resistant to kanamycin and streptomycin. Plasmids (mol. wt. 16.5 mega daltons) were isolated from five differentE. coli strains which harboured only a single plasmid and were characterized on the basis of antibiogram. Moreover, the transformation experiments were also performed to confirm the resistant character on the plasmid. We conclude that multiple drug resistance among most of theE. coli isolates is plasmid borne.  相似文献   

11.
Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.  相似文献   

12.
Pleural effusion is one of the commonest presentations of tuberculosis, the clinical manifestations being typically abrupt resembling bacterial pneumonia. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. Owing to these facts, tuberculous pleurisy as an extra-pulmonary disease poses a diagnostic dilemma. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in pleural fluid and are of limited use in diagnosis of tuberculous pleurisy. We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of tuberculous pleurisy by targeting the gene segment coding for MPB64 protein specific forMycobacterium tuberculosis. Based on the clinical criteria, 82 patients with lymphocytic exudative pleural effusion were included in the study. Patients were analyzed in two groups; one group consisting of 48 patients of tubercular pleural effusion confimed by various diagnostic procedures and another group of 34 patients comprising of non-tubercular pleural effusion. There were no false positive results by PCR and the specificity worked out to be 100%. Twenty two patients tested positive for Mantoux with a sensitivity of 45%. ZN-staining for AFB was found in samples from 15 patients (20% sensitivity). ADA was positive for 28 patients with a sensitivity of 53%. PCR was positive for 32/48 patients (67% sensitivity). Thus, PCR was found to be more sensitive than any other conventional method in diagnosis of clinically suspected tubercular pleurisy.  相似文献   

13.
After demonstrating that trifluoperazine (TFP) possesses invitro antitubercular activity against drug (single and multidrug) resistantMycobacterium tuberculosis, we initiated preliminary clinical studies in a few patients of tubercular lymphadenitis. Effect of TFP was assessed by testing the antitubercular activity of the serum of patients receiving TFP in addition to regular therapy. Patients were divided into two groups of 30 each. For ethical considerations, patients of both groups were treated initially for one month with antitubercular therapy (ATT) consisting of isoniazid, rifampicin, ethambutol and pyrazinamide and TFP was tried for 15 days only. Patients of group1 were given a single dose of TFP (5mg/day) daily from days 31 to 45 in addition to ATT, while those in group 2 received ATT only. Assessment of the antitubercular activity of the serum (testedin vitro in Youmans and Karlson’s liquid medium) revealed that the serum of patients (collected on 45th day) of group1 (ATT+TFP treated) possessed much higher antitubercular activity than that of group 2 (ATT only treated) patients. Clinical examination indicated that overall improvement was seen much earlier in group1 (ATT+TFP) patients than in group 2 (ATT alone) patients. At the end of the follow up period of 6 months with ATT from 46th day onwards to both groups, there were no side effects due to TFP. Hematology and liver function tests were normal in both the groups. We suggest that TFP has good potential and therefore deserves further studies either in combination with other drugs of ATT or as one of the drugs of ATT, for the treatment of tuberculosis due to MDR strains to find a suitable effective dose without side effects.  相似文献   

14.
The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 μS cm−1), almost double that of active cells (560±20 μS cm−1), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 μS cm−1) overall. It was also found that at 80 kHz, 900 μS cm−1 dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 μS cm−1 active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.  相似文献   

15.
High ferritin levels have been found to be associated with non infectious as well as infectious causes including tuberculosis. This is one case report of 41 year old male who presented with cough with expectoration. The patient had Multi drug resistant tuberculosis (MDRTB) and type 2 diabetes mellitus. The laboratory findings showed Iron 280 μg/dl, Total iron binding capacity (TIBC) 61 μg/dl, and ferritin 92,945 ng/ml which indicates that iron is an essential nutrient for the survival of the pathogen Mycobacterium tuberculosis.  相似文献   

16.
Based on our demonstration earlier that ethanol extract, water extract and a compound purified from garlic possessedin vitro antitubercular activity against drug resistant and susceptibleMycobacterium tuberculosis, we tried the effect of garlic extract in 30 patients of tubercular lymphadenitis. For ethical considerations, two groups of patients, 30 each, were given antitubercular therapy (ATT) consisting of isoniazid, rifampicin, ethambutol and pyrazinamide for 30 days. For the next 15 days (31 to 45 days) group 1 patients received 3–6 garlic pearls per day in addition to ATT while group 2 patients received ATT only. From 46th day onwards both the groups received ATT only for 6–8 months. Antitubercular activity of the serum samples collected on 45th day was assessed by its effect on the growth ofM. tuberculois. The serum of group 1 patients showed significantly much higher antitubercular activity than that of group 2 patients. Further, there was relief of dyspeptic symptoms caused by ATT therapy in patients of group 1 with garlic plus ATT therapy but no change in group 2 patients with ATT only. Liver function and hematological tests were normal in both the groups after 6 months of therapy. Garlic extracts or compounds have a good potential as antitubercular(s) drug if given as a supplement to ATT.  相似文献   

17.
Allicin was prepared from the ethanol extract of garlic without the use of heat. It inhibited the growth ofMycobacterium tuberculosis H37Rv andM. tuberculosis TRC-C 1193 resistant to isoniazid completely when grown as surface cultures as well as shake cultures. The minimum inhibitory concentration (MIC) was 70 μg/ml for both organisms. Further purification of allicin over silica gel-G columns gave a chloroform elutable fraction called CEF-allicin which inhibited the growth of both the susceptible and the isoniazid resistant strains ofM. tuberculosis. Its MIC is 25 μg/ml and is the lowest reported so far when compared with very high inhibitory concentration of 1 to 3mg/ml reported by other workers. It completely inhibited the synthesis of lipids and DNA from14C-acetate and3H-thymidine respectively and almost completely that of proteins and glycine derived carbohydrates from14C-glycine ofM. tuberculosis within 6 hr of exposure to CEF-allicin.  相似文献   

18.
Water extract of garlic (Allium sativum) inhibited the growth ofMycobacterium tuberculosis H37Rv andM. tuberculosis TRC-C1193 susceptible and resistant to isoniazid respectively. The minimum inhibitory concentration (MIC) was slightly above 80 but less than 160 μg/ml and slightly above 100 but less than 200 μg/ml for the susceptible and resistant strains respectively. Gel filtration in Sephadex G-100 columns showed that two protein fractions (43 & 38 kD) possessed antitubercular activity with much lower MICs of 20–40 μg/ml and 30–60 μg/ml for susceptible strain. Water extract, when added to actively growingM. tuberculosis in their mid log phase prevented their further growth. The water extract of garlic inhibited the incorporation of14C glycine into whole cells by 81% in 6 hrs. indicating that the primary mechanism of action is by inhibition of protein synthesis.  相似文献   

19.
BackgroundThe search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications.ResultsHere, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG–INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG–INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 μM. Similar trends are observed for 0.1, 1, and 5 μM.ConclusionsIn summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher anti-mycobacterial potency than the neat drug.How to cite: González-Torres M, Guzmán-Beltrán S, Mata-Gómez M, et al. Synthesis, characterization, and in vitro evaluation of gamma radiation-induced PEGylated isoniazid. Electron J Biotechnol 2019; 41. https://doi.org/10.1016/j.ejbt.2019.07.005.  相似文献   

20.
An 18kDa protein was identified as a major immunodominant allergen/antigen secreted by a wild type isolate and various clinical isolates ofA. fumigatus. The protein was purified to homogeneity and the N-terminal amino acid was found to be alanine. The N-terminal 20 amino acid sequence of 18kDa was found to be similar to restrictocin, a cytotoxin secreted byAspergillus restrictus. Mass spectroscopic analysis of the purified allergen revealed a molecular size of 17.01 kDa. Immunoreactivity of the purified allergen with monoclonal antibodies and specific IgG and IgE antibodies of the patients of aspergillosis confirmed that this protein is Asp fl.  相似文献   

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