Objective To study the mutation of DNA fragment of rpoB gene in different degrees of rifampin-resistance in Mycobacterium tuberculosis.Methods DNA fragment of rpoB gene in Mycobacterium tuberculosis was sequenced,including 32 low-level (R50) rifampin-resistant strains (50?g/mL rifampin-resistant),22 high-level (R250) rifampin-resistant strains (250?g/mL rifampin-resistant),10 (R0)rifampin-sensative strains and 1 H 37 Rv strain.Results No mutation was detected in 10 rifampin-sensative strains and 1 H 37 Rv strain;25(78.1%)rifampin-resistant strains had mutations in R50 and 21(95.5%)rifampin-resistant strains had mutations in R250(P=0.170).The mutatione points were distributed disorderly in R50.The 531-Ser mutation(57.1%)and joint mutation(23.8%)were more in R250 than those in R50.Conclusion The frequency of mutation in the rpoB gene of rifampin-resistant strain is higher.The mutation points are distributed disorderly in R50.The 531-Ser mutation(57.1%)and joint mutation(23.8%)are major mutative characteristics in R250.

We compared the MGIT 960 method,the reference,and the broth microdilution method for detecting the susceptibility of Mycobacterium tuberculosis isolates to protionamide (PTO).We performed drug susceptibility testing for 248 M.tuberculosis clinical isolates to PTO using MGIT 960 and broth microdilution method.In addition,a total of 117 isolates were randomly selected for further evaluation of the consistency of the minimal inhibitory concentrations determined by these two methods,and eleven concentrations of PTO had been involved accordingly (0.062 5,0.125,0.25,0.5,1,2,4,8,16,32,64 μg/mL).The MGIT method showed an average detection time of 10.1 days,while the detection period of broth microdilution method was 8 days,and the difference was statistically significant (P＜0.001).In addition,the rate of the sensitivity,specificity and concordance between these two methods was 96.5％ (55/57),93.2％ (178/191),and 94.0％ (233/248),respectively.The Kappa value was 0.84.Comparison of the MIC values detected by different methods revealed that the overall concordance rate was 81.2％ (95/117).For the isolates harboring low MIC values (MIC＜8.0 μg/mL),the concordance rate was 86.3％ (82/95),while that of the isolates with high MIC values was only 59.1％ (13/22).In conclusion,our data demonstrate that the broth microdilution method showed excellent concordance with MGIT method for detecting the resistance rate of M.tuberculosis isolates to PTO,indicating that the broth microdilution method with available performance,short turn-around time and convenient manual operation was suitable for rapid detection of M.tuberculosis to PTO.

Objective To study the feasibility of in vitro release of γ-interferon tests (IGRA) in screening of tuberculosis for children with close TB contacts. Methods 185 children with close TB contacts were detected by IGRA at the pediatric clinic in our hospital. Results In IGRA-positive group, the rate of strong positive PPD (PDD≥15 mm) was 50.9%, which was higher than 9.1% in IGRA-negative group (X2 =37.263, P < 0.00). The morbidity rate for children with close TB contacts was 30.2% in IGRA-positive group, and it was significantly higher than 3.0% in IGRA-negative group (X2 = 28.928, P < 0.00). The sensitivity was 80% and the specificity was 77.6% for IGRA screening in children who had close contacts with TB patients. The sensitivity would be 95.0%, as the test was combined with PPD test. Conclusions IGRA screening in children with close TB contacts can increase the detection rate of tuberculosis and reduce imaging screening.

ObjectiveTo learn the rpoB mutation difference in rifampicin/rifabutin cross-resistant (RIF/Rfb-R)clinical isolates and in rifampicin-resistant/rifabutin-susceptible (RIF-R/Rbo-S)clinical isolates of Mycobacterium tuberculosis.Methods To sequence the full-length genome of rpoB gene,and analyze the rpoB full-length gene mutation differences in 278 RIF/Rfb-R clinical isolates,40 RIF-R/Rfb-S clinical isolates,30 rifampicin-susceptible/rifabutin-susceptible (RIF-S/Rfb-S) and in 1 reference strain ofH37Rv.ResultsNo mutations of rpoB full-length gene were found in H37Rv reference strain and RIF-S/Rfb-S clinical isolates.In RIF/Rfb-R clinical isolates,531 (70.5％ ) and 526 (20.9％ ) were the most frequent mutation codons.223 (80.2％ ) isolates possesed single mutations as S531L,S531W,H526D,H526Y,H526R,Q513K,Q513P,Q510H,V176F,P287R,Y395C and H404Y.55 (19.8％) isolates had multiple mutations,and among these the S531L,H526 R,H526Y,H526D,D516G and Q513K were the main substitutions which were in combination with other points.In RIF-R/Rfb-S clinical isolates,516 (65.0％),526 ( 17.5％ ) and 533 ( 10.0％ ) were the most frequent mutation codons.21 (52.5％ ) isolates possesed single mutations as L533P,H526L,H526S,S522L,D516V,D516Y and D516F.19 (47.5％)isolates had multiple mutations and among these the D516V and L533P were the main substitutions which were in combination with other points.CondusionsIn our study,100％ rifamycin-resistant clinical isolates of Mycobacterium tuberculosis had rpoB mutations,but the mutations in RIF/Rfb-R clinical isolates were sharply different from RIF-R/Rfb-S clinical isolates in mutation positions or amino acids substitutions of single mutations strains,mutation positions or combination types and the most frequently mutation codons or amino acids substitutions of multiple mutations strains.Thus,DNA sequencing is instructive and meaningful to choose rifampicin or rifabutin for tuberculosis treatment.

Objective To detect the related genes with rifampin and isoniazid in Mycobacterium tuberculosis in sputums using the DNA chip technique and evaluate the fensibility of the clinical application of the DNA chip technique.Methods 586 sputum smear specimen was detected using the L-J cultivation to determine their drug resistance.Simultaneously.DNA chip was employed to detect the mutation of the frequent mutable points rpoB,katG/inhA in mycobaeterium tuberculosis isolates.These two assays were compared and samples showing discrepancy were chosen for additional sequencing to evaluate the accuracy of the detection.Results(1)There were 584 culture positive sputum smear specimens including 3(+)163 specimens,2(+)204 specimens,and 1(+)217 specimens.The drug fast results displayed that 361 strains were sensitive to INH,223 strains tolerated INH in which 93 strains tolerated it in low concentration while sensitive to it in high concentration.and 130 strains tolerated it in both low and high concentration.While 327 strains were sensitive to RFP.247 strains tolerated RFP in which 59 strains tolemted it in low concentration while sensitive to it in high concentration,and 188 strains tolerated it in both low and high concentration.(2)There were 367 positive strains(62.8%)and 217 negative strains(37.2%)identified by PCR amplification of the specific resistance gene fragments.The detection rate of the katG/inhA was 28.4%,and the mutation sites were mainly focused on the katG315(89.8%).The detection rate of the rpoB was 55.9%(137/247),and the mutation sites were mainly focused on rpoB531(68.6%)and rpoB 526(16.1%).(3)The sequencing of sample,which showed discrepancy with L-J cultivation and the DNA chip confirm a certain omission ratio.Conclusions It is feasible to detect the related resistant genes in Mycobacterium tuberculosis isolates using the DNA chip technique.The key factor is to raise the efficiency of the DNA extraction,the effciency of the PCR and the quality control of the experiment to facilitate its clinical application.

Objective To investigate the dynamic change and the clinical curative effect evaluation of plasma (1-3)-beta glucan D (BG) in the patients with pulmonary disease complicating fungal infection .Methods The MB-80 miroorganism dynamic rapid de-tection system and fungi BG detection kits were adopted to detect plasma BG content before and after treatment in 87 cases of pul-monary disease complicating fungal infection and the controls .The sputum culture in the patients was performed before and after treatment .Results Plasma BG levels before antifungal therapy ,at 1 ,2 weeks after treatment in 87 patients were (162 .81 ± 70 .03) , (15 .89 ± 30 .88) and (4 .58 ± 7 .87)pg/mL ,which in the control group was (5 .62 ± 1 .83)pg/mL ,plasma BG level had statistical differences between before treatment and at 1 ,2 weeks after treatment in the patients with the control group (P<0 .05);Plasma BG levels between at 1 week after treatment with at 2 weeks after treatment and the control group had statistically significant differ-ences (P<0 .05) .Among 87 patients ,66 cases were positive sputum culture at 1 week after antifungal drug treatment and 9 cases were positive sputum culture at 2 weeks after treatment .Conclusion Continuously monitoring the patient′s plasma BG level com-bined with the sputum fungal culture results ,clinical symptoms and lung shadow in X-ray has certain clinical value to judge the anti-fungal effect .

Objective To evaluate a rapid biochip system for the determination of muhidrugresistant tuberculosis (MDR-TB) in Mycobacterium tuberculosis isolates. MethodsA total of 1 186 clinical strains, including 800 rifampin (RFP) resistant isolates, 797 isoniozid (INH)resistant isolates, 791 MDR-TB and 380 susceptible strains, were selected from Beijing Chest Hospital, Shanghai Pulmonary Hospital and Guangzhou Chest Hospital respectively using stratified sampling method. Biochips were used to detect loci of rpoB 511 (T→C), 513 (A→C, C→A), 516 (G→T, A→T, A→G) , 526 (C→T, C→G, A→T, A→G), 531 (C→T, C→G), 533 (T→C), katG 315 ( G→C, G→A) and inhA -15 (C→T). Absolute concentration drug susceptibility test of RFP and INH were performed to serve as the gold standard to calculate susceptibility, specificity and overall concordance of biochip test. All polymerase chain reaction (PCR) products were sequenced to confirm the mutations. ResultsThe concordances between the biochip system and absolute concentration drug susceptibility test were 93.7％ ( 1 108/1 183 ) for RFP, 83. 8％(994/1 186) for INH and 82.4％ (975/1 183) for MDR-TB. Compared with absolute concentration drug susceptibility test, the biochip method displayed a sensitivity of 92. 0％ (733/797) and 77. 4％ (617/797)and a specificity of 97. 2％ (375/386) and 96. 9％ (377/389) for RIF and INH, respectively. For MDR-TB, the biochip system reached a sensitivity of 74. 6％ ( 588/788 ) and a specificity of 98.0％ ( 387/395 ).Among rpoB mutants, mutations were mostly detected at codon 531[64. 5％ (480/744)]. In stains with mutations in katG or inhA, 77.4％ ( 487/629 ) had mutation at codon 315 ( TCG ) of katG only. The sequencing results had a high concordance with that of the biochip method. There were slight differences in 5 strains, among which one strain was detected by biochip as katG 315(G→C) mutant, but was identified by sequencing as wild type, and mutation types other than those detected by the biochip were confirmed in the other 4 strains by sequencing. Conclusion This biochip system is adapted for extensive application in clinical diagnosis, as it allows fast and reliable detection of resistance to isoniazid and rifampin in tuberculosis clinical isolates.

Mycobacterium abscessus complex (MABC), a rapidly growing nontuberculous mycobacterium, has received increasing attention worldwide due to its rising isolation rate. The similarity of symptoms between MABC pulmonary disease and tuberculosis, different treatment methods required by different subtypes, as well as high levels of innate, adaptive and acquired antibiotic resistance, make MABC treatment more difficult and lead to unfavorable clinical outcomes of patients. This article reviews the basic characteristics, common antibiotic resistance mechanisms, as well as diagnosis and treatment of MABC, to provide reference for future research and clinical treatment of MABC lung disease.

Objective To study the clinical value of simultaneous amplification and testing for detection of Mycobacteria tuberculosis(SAT-TB)in sputum samples and bronchoalveolar lavage fluid(BALF)samples. Methods Totally 169 sputum samples and 151 BALF samples from suspected pulmonary tuberculosis patients were detected by both SAT and Bactec MGIT960.The sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of the samples using SAT-TB were calculated. Results Taken the results of BD960 as the reference,the sensitivity,specificity,PPV and NPV using SAT-TB of sputum samples were 84.00% (42/50),93.06%(67/72),89.36%(42/47)and 89.33%(67/75)respectively;and those of BALF samples 89.19% (33/37),95.12%(39/41),94.29%(33/35)and 42.39%(39/92)respectively.Taken clinical diagnostic results as reference standard,the sensitivity,specificity,PPV and NPV using SAT-TB of the sputum samples were 57.73% (56/97),93.06%(67/72),91.80%(56/61),and 62.04%(67/108)respectively;and those of BALF samples 51.82%(57/110),94.29%(39/41),96.61%(57/59)and、42.39%(39/92)respectively.The sensitivity,specificity, PPV and NPV using BD960 of the sputum samples were 51.55%(50/97),95.83%(69/72),94.34%(50/53),and 59.48%(69/116)respectively;and those of BALF samples 33.64%(37/110),90.24%(37/41),90.24%(37/41) and 33.64%(37/110)respectively.Conclusion SAT-TB is a rapid and sensitive method for the detection of Myco-bacteria tuberculosis in sputum and BALF samples.It can improve the detection rate of mycobaterium tuberculosis.