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Objective To investigate the correlation between the mutation of pncA gene and the susceptibility to pyrazinamide ( PZA) in Mycobacterium tuberculosis complex ( MTBC) strains and to analyze the mutation of panD and rpsA genes in wild type isolates without pncA gene mutation.Methods The sus-ceptibilities of 108 MTBC strains to first-line drugs including PZA were detected by using the MGIT 960 TB system.PCR was performed to amplify the 16S rDNA and pncA, panD and rpsA genes.The PCR products were analyzed by DNA sequencing analysis .Results Among the 78 multidrug-resistant MTBC strains , 47 isolates (60%) were resistant to PZA.Four out of 30 (13%) strains that were sensitive to ethambutol , iso-niazid, rifampicin and streptomycin (EIRS) were resistant to PZA.The drug-resistant MTBC strains showed higher resistance rate to PZA than that of the EIRS sensitive strains .There were 49 ( 96%) PZA-resistant isolates and 4 (7%) PZA-sensitive isolates occurred pncA gene mutation.Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation , especially the His57Asp substitu-tion.The pncA gene mutations centralized in the regions of 160-169, 203-289, 309-396 and 413-467.Seven novel mutation sites of pncA gene were observed including T175C, C188A, G insertion at 68, AGC insertion at 235, C insertion at 339, CC insertion at 392 and GT deletion at 395.The mutation sites founded in the genomes of PZA-sensitive strains were different from those of the PZA-resistant strains .No mutation of the pncA gene and the upstream regulatory sequence was found in two PZA-resistant strains , NJ44 and NJ108 . The sequence analysis of panD and rpsA gene showed that the NJ 108 strain had panD gene mutation at G419A, but no mutation was detected in the NJ 44 strain.Conclusion The multidrug-resistant MTBC strains showed higher resistance rate to PZA .The pncA gene mutation was common in PZA-resistant MTB strains and the panD gene mutation was also worthy of attention .
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[Abstract] Objective To evaluate the effect of Mycobacterium tuberculosis Direct Assay (MTD) for rapid detecting Mycobacterium tuberculosis rRNA and Multi-locus PCR for M.bovis BCG strain typing in patients with suspected extra-pulmonary tuberculosis.Methods From June 2010 to December 2011,47 children and 75 adult patients with suspected extra-pulmonary tuberculosis in Shanghai public health clinical center were recruited.Also 48 non-tuberculosis patients were taken as a negative control.Clinical specimens from these patients were collected.Acid fast stain,solid culture,liquid culture,and MTD were used to detect all clinical specimens simultaneously.Screen tuberculosis strains of the culture isolates by MPT64 antigen assay and use Multi-locus PCR for the BCG strain genotyping of the isolates without MPT64 antigen.SPSS16.0 was used to analyse the results.Results The sensitivity for acid fast stain,solid culture,liquid culture and MTD test was 10.7％ (13/122),11.5％ (14/122),16.4％ (20/122) and 37.7％ (46/122),respectively.And the specificity of MTD was 100.0％.Six clinical isolates from children were identified as BCG by Multi-locus PCR typing,the same with chemical tests.Conclusions The MTD assay and the MGIT960 liquid culture are effective and reliable method for diagnosing extra-pulmonary tuberculosis.And Multi-locus PCR can be assisted for the early diagnosis of extra-pulmonary tuberculosis patients with suspected BCG infection.
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ObjectiveTo analyze the characterstics of phenotype and genotype of multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) by molecular line probe assay and liquid culture with MGIT960.MethodsGenoType MTBDR Kits were used for identifying the types of the first-line and second-line antituberculosis drug resistant genes partly and BD MGIT960 was used for detecting the chug susceptibility.Results( 1 ) Out of 94 MDR-TB strains,the rate of drug resistant to EMB,AMK,OFX and MFX by BD MGIT960 assay were 36.2%,17.0%,54.3% and 55.3%,respectively.Among these isolates,13 were extensively drug resistant tuberculosis (XDR-TB).(2) Compared with MGIT960,the concordance rate of GenoType MTBDRplus was 86.2% and 95.7% respectively.Taking MGIT960 results as reference,the sensitivity of GenoType MTBDRsl detecting the susceptibility of EMB,AMK,OFX and MFX to 94 isolates were 47.1%,81.3%,94.1%,94.2%,respectively.The specificity were 75.0%,98.7%,90.7%,92.9%,respectively.(3) Among the rpoB mutation categories,S531L accounts for most.MTB resistant to IFN caused by the mutation of katG chiefly and the S315T1 was in the majority.The gyrA mutation sites located at the ninety-fourth codon most.Out of 94 strains,23 were mixed with 2 kindsof Mycobacterium tuberculosis at least and 7 were undetectable mutations.Conclusion Among the M/XDR-TB,the strains resistant to INH,RFP,AMK,OFX and MFX were caused most by the mutation of katG,rpoB,rrs and gyrA,respectively.The relationship between EMB and embB was not so clear relatively.As a fast detecting drug susceptibility test kit,GenoType MTBDR possess good sensitivity and specificity.So,it could be as an assistant method to guide the therapy on clinic.