Clinical diagnostic performance of the simultaneous amplification and testing methods for detection of the Mycobacterium tuberculosis complex for smear-negative or sputum-scarce pulmonary tuberculosis in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Early detection of pulmonary tuberculosis (PTB) is a big challenge in smear negative and sputum scarce patients in China. Simultaneous amplification and testing methods for detection of the Mycobacterium tuberculosis (MTB) complex (SAT-TB assay) is a novel molecular technique established in our hospital. This method has a high sensitivity and specificity in the lab. In this study, the clinical diagnostic performance of this method in smear-negative or sputum-scarce PTB suspects was investigated and evaluated.</p><p><b>METHODS</b>Two hundred smear negative and 80 sputum-scarce patients were recruited in this study. Samples that included sputum or bronchial washing fluid were collected and sent for both bacteria culture and SAT-TB assay. Diagnosis for these patients was based on the comprehensive evaluation of chestX- ray/CT study, histology examination, lab results, and treatment response. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were investigated and calculated using confirmed tuberculosis (TB) and non-TB cases. The time required for detection of MTB was also measured for each method.</p><p><b>RESULTS</b>Ninety-two patients (33%) were diagnosed as definitive TB, 112 patients (40%) were probable PTB, and 76 (27%) were non-TB. The sensitivity, specificity, PPV, and NPV of SAT-TB in smear-negative PTB suspects were 93% (95% CI, 84%-98%), 98% (95% CI, 90%-100%), 98% (95% CI, 91%-100%), and 93% (95% CI, 83%-98%). In sputum scarce PTB suspects, the sensitivity, specificity, PPV, and NPV of the SAT-TB assay on bronchial washing fluids were 90% (95% CI, 74%-98%), 100% (95% CI, 85%-100%), 100% (95% CI, 88%-100%), and 88% (95% CI, 69%-97%). The accuracy of the SAT-TB assay is consistent with the bacteria culture assay. The median time required for detecting MTB in the SAT-TB assay was 0.5 day, which was much faster than bacteria culture (28 days).</p><p><b>CONCLUSIONS</b>The SAT-TB assay is a fast and accurate method for the detection of MTB. It can be widely applied in the clinic and be an asset in early detection and management of PTB suspects, especially in those patients who are smear negative or sputum scarce.</p>

Induction in vitro and stability of Mycobacterium tuberculosis resistance to ofloxacin / 中华预防医学杂志

<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>

Expression and clinical application of recombinant fusion protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosts / 中华检验医学杂志

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.

An analysis on 200 randomized clinical isolates of Mycobacterium in Sichuan / 中华微生物学和免疫学杂志

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.

Research on the association of DC-SIGN gene promoter polymorphisms with tuberculosis / 中华微生物学和免疫学杂志

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.

Evaluation of Microscopic Observation Drug Susceptibility Assay in Detection of Mycobacterium tuberculosis / 中华医院感染学杂志

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.

The study of bacteriophage based assay detecting Ethambutol resistance in clinical isolates of Mycobacterium tuberculosis / 中华传染病杂志

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.

Study on simultaneous detection of four drugs resistance anti-tuberculosis front line by phage amplified biologically assay / 中华传染病杂志

Objective Evaluating possibility of phage amplified biologically(PhaB) assay in detecting susceptibility of Mycobacterium tuberculosis(MTB) to four first-anti-tuberculosis-drugs on the same time and of 139 clinical isolates to streptomycin(S), isoniazid(H), rifampin(R) and ethambatal(E) at the same time, comparing with the results of Bactec-960 and determining the minimal inhibitory concentrations(MIC) of isolates which results were not consistent. Results Concordance rates of the susceptibility to S, H, R and E in 139 clinical isolates detected by PhaB and Bactec-960 are 97.1%, 99.3%, 95.7% and 95.0% respectively. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, positive and negative predictive value (PPV and NPV) as well as accuracy of susceptibility test to S detected by PhaB assay was 90.0%, 99.1%, 96.4%, 98.2% and 97.1% respectively, to H 97.9%, 98.9%, 97.9%, 98.9% and 95.0% , to R 86.2%, 97.3%, 89.3%, 96.4% and 95.0%, to E 81.0%, 97.5%, 85.0%, 96.6% and 95.0%. There are 19 inconsistent results of 13 isolates in comparing PhaB with Bactec-960. 18 results of 12 isolates by MIC are identical with the results of PhaB assay. 1 result of 1 isolate is identical with Bactec-960. Conclusions[KG1]The results of susceptibility to S, H, R and E detected by PhaB were highly concordance rate with the results of Bactec-960. PhaB assay can be used for rapid screening of susceptibility test for MTB.

Comparison of four kinds of method in detection isoniazid resistance in clinical isolates of mycobacterium tuberculosis / 中华传染病杂志

Objective To establish Phage Amplified Biologically Assay (PhaB) detecting isoniazid(INH) resistance rapidly and evaluate PhaB assay for drug susceptibility testing of isoniazid(INH) in clinical isolates of Mycobacterium tuberculosis(MTB). Methods Detecting the INH resistance of 167 clinical isolates of MTB by PhaB assay,comparing the results of PhaB with that of Bactec-960 system and analyzing the sensitivity, specificity and accuracy of PhaB assay. Results When the mixture of 0.2 ?g /ml INH and MTB was incubated in 37℃ for 48 h, the accurate results were obtained rapidly by calculate the reduce of plaque of PhaB assay. If the results of Bactec-960 system is the golden standard, the sensitivity, specificity, PPV, NPV and accuracy of PhaB assay was 96.4%, 96.4%, 93.1%, 98.2% and 96.4% respectively. Conclusions The PhaB assay with highly sensitivity specificity are highly consistent with Bactec-960 system. Not only it takes only three days to detect drug susceptibility of INH in clinical isolates of MTB but also it is easily to operate. We believe that this low-cost assay may be a good rapid screening of INH resistance in MTB isolates.