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Objective To establish a method of judge the pyrazinamide(PZA) susceptibility for Mycobacterium tuberculosis(MTB) with 24-hole micro-liquid culture silica gel color plate,and evaluate the clinical application value of this method.Methods According to the result of MGIT960,to detect PZA drug susceptibility for 30 MTB clinical isolates of whose PZA susceptibility were known with silica gel color plate.The effects of different pH value,different inoculation concentrations on the results were observed,and the optimum detection conditions were discussed.Finally,the 98 MTB clinical isolated of whose PZA susceptibility were unknown were simultaneously detected by gel color plate and MGIT960,the sensitivity,specificity and accuracy were judged by PZA drug sensitivity.Results The best pH was 5.8-5.9 and the best concentration was 2.5×10-1 mg/mL in gel color plate.The best results were read after 7-14 d.The sensitivity,specificity and accuracy were 95.50%,96.30%,and 98.21% respectively just at PZA critical concentration was 100 μg/mg.The sensitivity,specificity and accuracy were 90.90%,92.59%,and 91.84% respectively just at PZA critical concentration was 200 μg/mg.Conclusion The PZA susceptibility for MTB with 24-hole micro-liquid culture silica gel color plate is accurate,rapid,low-cost and simple.
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Objective To study the effect of microscopic observation drug susceptibility assay (MODS)for testing Mycobacteri-um tuberculosis in sputum ,and to evaluate its clinical value .Methods A total of 150 sputum samples were collected from patients with pulmonary tuberculosis ,and Mycobacterium tuberculosis were detected by Lowenstein-Jensen (L-J) method and MODS ,and compared the results of the two methods .Results The concordance rates of the test results of sputum sample between MODS and L-J was 77 .8% .The sensitivity and specificity of MODS were 93 .3% and 70 .0% ,positive predictive value (PPV ) was 60 .8% , negative predictive value (NPV) was 95 .4% ,as well as accuracy by MODS was 77 .8% .Conclusion MODS assay could be used as a rapid tuberculosis detection method ,with rapid ,sensitive ,simple ,and other advantages .
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Objective:To study the in vitro antimicrobial activity of traditional Chinese medicine herbal pair of euphorbia humifusa Willd. and portulaca oleracea L. . Methods: The antibacterial effect in vitro of the traditional Chinese medicine herbal pair and the single herb of euphorbia humifusa Willd. and portulaca oleracea L. was studied on staphylococcus aureus, escherichia coli and pseudo-monas aeruginosa by the 2-fold dilution method and the broth micro-dilution method in a 96-well plate. The minimum inhibitory con-centration ( MIC) , minimum bactericidal concentration ( MBC) and the diameter of inhibition zone were determined. Results:The ex-perimental strains showed the different sensitivity among the traditional Chinese medicine herbal pair and the single herb. The antibac-terial and bactericidal activity of the traditional Chinese medicine herbal pair was the most obvious(P<0. 05). As the temperature in-creasing, the antibacterial activity of all water extract on different experimental strains changed. The results of MIC and MBC showed that the effects of water extract on escherichia coli were strongest, that of stapphylococcus aureus were secong, ant that of pseudomonas aeruginosa were relatively weak. Conclusion:The traditional Chinese medicine herbal pair of euphorbia humifusa Willd. and portulaca oleracea L. has antibacterial and bactericidal activity in varying degrees on the experimental strains with some differences, and the changes in the application forms of traditional Chinese medicines has great influence on the antibacterial and bactericidal ability.
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<p><b>OBJECTIVE</b>To induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.</p><p><b>METHODS</b>MTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.</p><p><b>RESULTS</b>MIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.</p><p><b>CONCLUSION</b>MTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.</p>
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Antitubercular Agents , Pharmacology , DNA Gyrase , Genetics , Drug Resistance, Bacterial , Genetics , Microbial Sensitivity Tests , Mycobacterium tuberculosis , Genetics , Ofloxacin , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.</p><p><b>METHODS</b>Inactive Mycobacterium tuberculosis reference strain H37Rv was used as the target molecule to screen the Ph. D.-C7C peptide library, and Mycobacterium bovis, BCG was used for reverse screening. After 4 rounds of affinity screening, single phages eluted by H37Rv and BCG were selected for DNA sequencing. ELISA was used to detect the binding affinities of different single phage clones. The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers. Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.</p><p><b>RESULTS</b>After 4 rounds of biopanning, phages that could bind with target molecules were remarkable enriched. 16 common sequences were obtained by sequencing analysis of single phages. With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H37Rv and BCG, the ratio to negative control of which were ≥ 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the positive clones. Based on the results of flow cytometer detection, the affinities to H37Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6 ± 4.5)%, (10.3 ± 1.8)%, (86.6 ± 7.9)%, separately, which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%) . From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, SB26 could bind to H37Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H37Rv, and didn't bind to 3 non-mycobacteria.</p><p><b>CONCLUSION</b>Based on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.</p>
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Base Sequence , Enzyme-Linked Immunosorbent Assay , Ligands , Mycobacterium tuberculosis , Peptide Library , PeptidesABSTRACT
Objective To evaluate the accuracy of pyrosequencing for the mutation detection of katG gene in isoniazid resistance in Mycobacterium tuberculosis using Meta-analysis.Methods Searching PubMed,Web of Science,Elsevier,and China National Knowledge Infrastructure (CNKI),Weipu and WANFANG DATA to obtain the relevant English and Chinese-language articles,respectively.A written protocol and explicit study selection criteria was followed.Quality of included trials was assessed by QUADAS (quality assessment of diagnostic accuracy studies).Subsequently,the characteristics of the included articles were appraised and extracted.Heterogeneity of the included articles was tested by using STATA 10.0,which was used to select proper effect model.The fixed effects model was adopted using Meta-Disc software.Finally,sensitivity analysis was performed.Results Totally 114 research papers were collected and 9 articles were selected.The accordance between pyrosequencing and conventional sequencing result was 100％.Eight studies were involved including 945 specimens when katG gene was detected.The overall sensitivity and specificity were 0.77 (0.73,0.80) and 1.00 (0.99,1.00),respectively.The area under the SROC was 0.9882.As inhA gene was detected,the overall sensitivity and specificity were 0.19 (0.15,0.24) and 1.00 (0.98,1.00).The test was stable.Conclusions Our meta-analysis reveals that pyrosequencing is a highly specific tool for detection mutation of katG gene of isoniazid resistance.This result suggests that it is useful for screening of isoniazid resistance in diagnostic test.(Chin J Lab Med,2013,36:329-332)
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Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue.Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results The expression plasmid pET32a-Rv0091 was constructed and expressed in E.coli.BL21trxB,and the optimum expression system was conformed.The purity of the recombinant protein was more than 95％.Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins.Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same.Enzyme assay showed the recombinant protein able to catalyze the substrate MTA.Enzymatic properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.
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Objective To find out the resistant situation and drug of Mycobacteria patients in Sichuan and offer foundation for clinical.Methods Two hundred randomized clinical isolates of Mycobacterium were determined by Roche drug sensitivity and minimum inhibitory concentration (MIC) method.Results Of the 200 clinical isolates,192 stains were Mycobacterium tuberculosis(MTB) (96.0％),8 strains (4.0％) were non-tuberculosis mycobacterium(NTM).Of the 192 MTB strains,108( 57.3％ ) sensitive strains and 84 (43.7％)stains were resistant to one or more than one drugs.Among these 84 resistant strains 23 were multi-drug resistant ( MDR,12.0％ ),4 were extensively drug resistant( XDR,2.1％ ).The anti-TB drug resistance rates were:SM(16.7％),INH(20.8％),RFP(17.2％),EMB(10.9％),PI(16.1％),LFX(8.8％),AMK ( 16.7％ ),CPM ( 6.2％ ),PTA ( 33.3％ ),respectively.Conclusion The resistance rate of tuberculosis keeps at a high level in Sichuan,especially the resistance rate of multiple (≥4) drug,we should oar attention.
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Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.
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Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.
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Objective To evaluate clinical application of phage amplified biologically assay (PhaB) in susceptibility test of Mycobacterium tuberculosis (MTB) in sputum. Methods The drug susceptibility of MTB was detected by PhaB in 143 patients with sputum-positive pulmonary tuberculosis (PTB),and the chemotherapy regimens were adjusted according to the results of susceptibility test.Independent samples t-tests were used for comparison of means.Count numbers were compared with Chisquare test.If there were count number of 0,Fisher probabilities should be used.ResultsThe total positive rate of PhaB was 94.4％ (135/143) with no differences between three types of PTB (x2 =1.886,P ＞ 0.05 ).The duration of testing for PhaB group was (6.6 ± 1.8) days,while for control was (29.4 ±8.7) days (t =29.01,P ＜ 0.01 ).Compared with control group,the 2-month negative-conversion rate (63.2％ vs.35.1％,x2 =3.989,P ＜ 0.05 ) and cure rate ( 100％ vs.78.4％,P ＜ 0.05 ) of PhaB group in type Ⅱ patients were significantly higher.But there were no differences between PhaB and control groups in type Ⅰ and Ⅲ PTB patients.ConclusionThe results of PhaB drug susceptibility test can be helpful for choosing effective chemotherapy regimen for PTB patients rapidly.
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Objective To test the drug susceptibilities in Mycobacterium tuberculosis by microscopic observation drug susceptibility(MODS)and evaluate the method for the detection susceptibility in second-line drugs.Methods To set up the MODS method.the drug susceptibilities of 4 second-line drugs in Mycobacterium tuberculosis were tested in 24 well plates.The test conditions were discussed.The resistance to protionamide(PTH),amikacin(AMK),capreomycin(CPM)and levofloxacin(LVF)of 60 MTB isolated from 2007 to 2008 in Shanghai Pulmonary Hospital were detected and compared with that of L-J method.The isolates were tested by minimum inhibitory concentration(MIC)method when the drug susceptibilities were not consistent.Results Among the 60 Mycobacterium tuberculosis clinical isolates.the results of drug susceptibility were confirmed by MODS,absolute concentration method and the accordance rate of PTH,AMK,CPM and LVF were 96.7%(58/60),98.3%(59/60),91.7%(55/60)and 96.7%(58/60),respectively.If the result of absolute concentration method was as the standard.the sensitivity.specificity.positive and negative predictive value as well as accuracy were 100%,87.5%,87.5%,100%and 96.7%in PTH;100%,90.0%,90.0%,100%and 98.3% in AMK;76.9%,95.7%,83.3%,93.8%,91.7% in CPM;100%,96.0%,83.3%,100% and 96.7% in LVF respectively detected by MODS assay.Conclusion MODS is a rapid and simple method for susceptibility testing of second-line drug in Mycobacterium tuberculosis.
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Objective To study the relationship of two variants( -871A/G and -336A/G) polymorphisms of the DC-SIGN gene with the susceptibility to pulmonary tuberculosis in Chinese population.Methods Two hundred and thirty-seven tuberculosis cases and 244 controls were genotyped by pyrosequencing in this case-control study. The analysis of the relationship of the -871A/G and -336A/G polymorphisms with their susceptibility of pulmonary tuberculosis(PTB) and the relationship of the two variants with their clinical correlation of tuberculosis was performed by chi-square test. Results The genotypic frequencies of A/G + G/G and A/A of - 871, 37.6%, 62.4% respectively in cases, and 43.4%, 56. 6%respectively in controls, had no significant difference in statistics. And the genotypic frequencies of A/G + G/G and A/A of -336, 12. 2% ,87.8% respectively in cases, and 14.3% ,85.7% respectively in controls, had also no statistical difference between two groups. Interestingly, a significant association is disclosed between the promoter variant - 336G allele and fever in patients ( P = 0. 037, OR = 0. 191, 95 % CI:0. 040-0. 907 ). Conclusion The single nucleotide polymorphism of -871A/G and -336A/G in DCSIGN gene promoter might not be associated with the susceptibility to tuberculosis in Chinese. Tuberculosis patients with -336G allele are significantly protected fever.
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Objective To investigate correlation between the results of drug susceptibility and the extent of drug-resistances in Mycobacterium tuberculosis clinical isolates. Methods Liquid culture and MTT test were used. Twelve anti-TB drug MICs and drug susceptibility testing of the 163 MTB strains from random clinical isolates were detected, which including RFP, INH, SM, EBM, OFLX, LVFX, MOX, AMK,CPM, PTA, CLA and PAIN. Results There are 67% (42/62) Mycobacterium tuberculosis strains resistant to SM, 63% (51/81) Mycobacterium tuberculosis strains resistant to INH, 77% (50/65) Mycobacterium tuberculosis strains resistant to RFP, 41% ( 15/37 ) Mycobacterium tuberculosis strains resistant to AMK,41% (12/29) Mycobacterium tuberculosis strains resistant to CPM, 20% (12/60) Mycobacterium tuberculosis strains resistant to EMB and 43% (25/58) Mycobacterium tuberculosis strains resistant to OFLX which MICs were equal to or more than 16 μg/ml, 8 μg/ml, 8 μg/ml, 16 μg/ml and 4 μg/ml, 4 μg/ml and 8 μg/ml,respectively. There were significant differences in the MICs of OFLX, LVFX and MOX in OFLX resistant strains (2-128, 1-32 and 0.0625-1 μg/ml, respectively) by ANOVA ( F = 16.874, P ＜ 0.001 ). The MICs of SM, INH, RFP, EMB, OFLX, AMK and CPM in isolates resistant to six or seven drugs (0.5-128,2-64,0.25-128,1-32,1-64,0.5-128 and 1-128 μg/ml,respectively) were higher than those (0.25-128,0.0625-64,0.25-32,0.25-2,0.125-2,0.5-4 and 1-4 μg/ml,respectively) in isolates resistant to one or two drugs (F=20.066, 40.499, 47. 197, 70.373, 91.432, 41.840 and 21.547, respectively, P ＜0.05). The MICs of SM, INH, RFP and EMB in isolates resistant to four drugs (1-128,2-64,0.25-128 and 1-32 μg/ml,respectively ) were higher than those ( 0.25-128,0.0625-64, 0.25-64 and 0.25-2 μg/ml,respectively) in isolates resistant to one or two drugs (F = 26.242, 23.563, 31.541 and 64.469,respectively, P ＜0.05).The MICs of RFP in MDR isolates (2-64 μg/ml) were higher than those (0. 25 μg/ml) in other resistant isolate except M DR isolates (F = 5.613, P <0.05). Conclusions The study shows that there are associations between the results of routine drug susceptibility testing and the resistant extent of anti-TB drugs. This could help doctors select more effective anti-TB regimen for TB patients according to the correlations.
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OBJECTIVE To set up and evaluate the microscopic observation drug susceptibility assay(MODS)technology and use it to detect rapidly Mycobacterium tuberculosis(MTB).METHODS The 24-hole cell culture plate method of liquid culture were used to set up MODS technology.The MODS technology was used to detect MTB and non-M.tuberculosis(NMT)isolates comparing with Lowenstein-Jensen(L-J) method.RESULTS When bacterial concentration was 3?103 CFU/ml,the time of reading test-result was the seventh day by MODS;four kinds of NMT(M.phlei,M.kansasii,M.chelonaeand M.marinum) in the liquid medium in the observation was similar to the growth of cording,it was difficult to distinct from the cording of MTB in the liquid medium;When using the 4-Nitro-benzoic acid 800 ?g/ml,thiophene-2-carboxylic acid hydrazine 2.5 ?g/ml for testing conditions,the correct diagnosis can be improved;the test results of clinical isolates by MODS were highly concordance rate with the results of L-J.If the results of L-J was the golden standard,the sensitivity,specificity,positive and negative predictive value as well as accuracy by MODS was 95.7%,100%,100%,99.9% and 97% respectively.CONCLUSIONS The results of MODS in detection of MTB are highly concordance wih the results of L-J method;MODS assay can be used for rapid detection of tuberculosis,with a rapid,simple,inexpensive,and other advantages.
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Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX(systematic evolution of ligands by exponential enrichment)technology. Methods A random ssDNA library with in vitro synthesized 78 nucleotides in length was subiected to 12 rounds of selection by SELEX method against MPT64 protein. The binding ability of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. Results The selective system used was as follows:in PCR amplification,annealing temperature was 65℃ and the concentration of Mg2+ was 1.5 mmol/L in optimizing library, and when preparation of ssDNA with asymmetrical PCR amplification, 0.75 mmol/L of Mg2+ was used. When using the plate for ELISA as the substrate for the selection, the pattern of electrophoretic band of PCR product after the tenth round selection became unitary and denser than that of the first round. The binding assay demonstrated that A value at 450 nm of the tenth round increased by 9.18 times as compared with that of the first round. The results showed that the affinities of the aptamers were different. The highest A value at 450 nm was 1.606, and the lowest 0.572. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein are successfully picked out from the initial random DNA library.
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<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of integrative Chinese and Western medicine in treating mid-advanced crescentic nephritis (MACN).</p><p><b>METHODS</b>Thirty-two patients, their diagnosis was confirmed as MACN by renal biopsy, were divided, adopting randomized, controlled method, into two groups, the treated group and the control group, they were all, excepting one, treated with impact therapy of methyl-prednisolone followed with oral intake of prednisone, to part of them cyclophosphamide or mycophenolate mofetil was given in addition, to those with hypo-hemoglobin (< 90 g/L), subcutaneous injection of erythropoietin was administered. Decoction of Qingre Huoxue recipe (QHR), consisted of oldenlandia herb 30 g, honey-suckle stem 30 g, violet herb 30 g, red peony root 15 g, rehmannia root 15 g, solomonseal rhizome 15 g, asiabell root 30 g, red sage root 30 g, prepared rhubarb 12 g and giant-hyssop herb 12 g, were additionally given one dose per day to patients in the treated group. The renal function, improvement of anemia and immunosuppressive agents needed in patients were observed after 3 months treatment.</p><p><b>RESULTS</b>After treatment, renal function was improved in both groups, but the effect in the treated group was better than that in the control group (P < 0.05). Anemia was partially alleviated in the two groups with no significant difference. The dosage of glucocorticoids used in the treated group was obviously lesser than that used in the control group (P < 0.01).</p><p><b>CONCLUSION</b>Integrative Chinese and western medicine could treat crescentic nephritis to obtain good effect, and reduce the quantity of glucocorticoid necessity for treatment.</p>
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Adult , Female , Humans , Male , Middle Aged , Drug Therapy, Combination , Drugs, Chinese Herbal , Therapeutic Uses , Glomerulonephritis , Drug Therapy , Immunosuppressive Agents , Therapeutic Uses , Kidney Function Tests , Kidney Glomerulus , Kidney Tubules , Methylprednisolone , Therapeutic Uses , PhytotherapyABSTRACT
Objective To set up and evaluate the method of Phage Amplified Biologically Assay (PhaB) in the rapid detection of detection of Ethambutol resistance. Methods To detect the EMB resistance of 102 clinical isolates of Mycobacterium tuberculosis by PhaB and compare it with the results of absolute concentration method. The minimum inhibitory concentration (MIC) was detected for all discrepancy isolates. Results Of all 102 strains in Mycobacterium tuberculosis clinical isolates, 82 strains were EMB susceptible and 20 strains were EMB resistant by PhaB method, while 77 strains were EMB susceptible and 25 strains were EMB resistant by absolute concentration method. 74 of 102 strains were EMB susceptible and 17 strains were EMB resistant by both methods. The concordant isolates of determination of EMB resistance were 91 strains in two methods and the concordance rate was 89.2%. There were 11 disconcordant isolates and the discrepancy rate was 10.8%. In the 11 strains of discrepant isolates between two methods, 7 strains (63.6%) were in accord with the results of MIC method (5 of 7 strains were EMB susceptible by PhaB but EMB resistant by the absolute concentration methods, 2 of 7 strains were EMB resistant by PhaB but EMB susceptible by the absolute concentration method). Conclusions The PhaB assay can be used for detection of EMB resistance in isolates of Mycobacterium tuberculosis easily and quickly within three days.This method do not need special instrument and may be used for rapid screening of M.tuberculosis with resistance to EMB.
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Objective To elucidate the relationship between polymorphisms of NRAMP1 gene and susceptibility to tuberculosis in Chinese Han nation by case control study. Methods We selected 127 patients with active pulmonary tuberculosis who were all Han people with mean age of 52.5 years and 58 ethnically matched healthy controls. We typed the polymorphisms of NRAMP1, INT4 and D543N, through polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) techniques. Results Two NRAMP1 polymorphisms were both significantly associated with smear positive pulmonary tuberculosis. Subjects who were heterozygous for polymorphisms in INT4 and D543N were related with tuberculosis. It was the cooperation of different polymorphisms of NRAMP1 that led to the susceptibility to tuberculosis. Conclusions The polymorphisms in NRAMP1 gene affect susceptibility to tuberculosis in Han people in China.
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Objective To develop a new Multiplex Allele-Specific polymerase chain reaction(MAS-PCR) assay to detect the main point mutations in the katG and rpoB genes, which has been reported to account for the majority of clinical Mycobaterium tuberculosis resistant to isoniazid and rifampin. Methods Based on the sequences of katG and rpoB genes, specific primers were designed to carry out the MAS-PCR to detect the most common mutations in codon315 of katG and codons 531,526,516 of rpoB gene. Results The purified DNA preparation of 96 clinical Mycobacterium tuberculosis strains were used to optimize PCR. No mutation was detected in 19 isoniazid-sensitive strains. The 315Ser point mutation was detected in 79.2%(61/77)of isoniazid-resistant strains, the type of mutation includes the most common S315T and the less common S315N, which could’t be detected by PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism). However, S315G couldn’t be detected by MAS-PCR and that will make a false negative. The mutations in codons 531,526,516 were detected by the MAS-PCR. Compared with the results of direct sequencing of rpoB gene, no mutation was detected in sensitive strains. For rifampin-resistant strains, the total sensitivity was 81.5%(66/81). Conclusions MAS-PCR is a new molecular method with high sensitivity and specificity, which can be used to detect the point mutation in katG and rpoB gene rapidly and economically. It can be used in clinical laboratories to detect drug-resistant tuberculosis strains. Simultaneous detection for katG and rpoB gene mutations in one MAS-PCR system will help to improve the efficiency of this method.