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Objective To investigate the species distribution characteristics of nontuberculous mycobacteria(NTM)in acquired immunodeficiency syndrome(AIDS)patients in Shanghai,and to provide evidences for clinical treatment.Methods A total of 775 Mycobacteria strains were isolated from patients(including 129 isolates from AIDS patients and 646 isolates from HIV-negative patients)admitted to Shanghai Public Health Clinical Center during 2015.All the species were identified by the sequence analysis of 16S rRNA gene and hsp65 gene.Differences in the species distribution were compared between patients with and without HIV infection.CD4+T lymphocyte count was detected by flow cytometry and its relation with mycobacteria infection was also analyzed.Chi-square test was used for comparison between groups.Results The ratio of NTM isolation from HIV-negative patients was 15.79%(102/646),while that was 46.51% in AIDS patients(60/129),and the difference was statistically significant(x2=61.38,P <0.01).Among the 60 NTM strains isolated from AIDS patients,mycobacterium avium-intracelluare complex(MAC)and Mycobacterium gordonae were the predominant species(43.33% and 20.00%,respectively).Moreover,five Mycobacterium colombiense strains,which were relatively rare,were also obtained.A total of 102 NTM were identified from HIV-negative patients,of which Mycobacterium gordonae(32 isolates,31.37%)and MAC(30 isolates,29.41%)were the most frequently isolated.In addition,the positive rates of NTM and MAC were significantly higher in patients with CD4+T lymphocyte counts ≤50 cells/μL(58.33% and 76.92%,respectively)than those with CD4+T lymphocyte counts>50 cells/μL(20.00% and 15.38%,respectively)(x2=4.048 and 6.524,respectively,both P<0.05).Four out of five Mycobacterium colombiense infected patients died of disseminated infections,except the remaining one whose CD4+T lymphocyte counts>50 cells/μL.Conclusions The prevalence of NTM isolation is significant higher in AIDS patients than HIV-negative patients in Shanghai,and the most prevalent NTM species is MAC.The NTM infection in AIDS patients is related with low CD4+T lymphocyte counts.
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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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Objective To improve the diagnosis of tuberculosis ( TB) by analyzing Mycobacterium infection in fine-needle aspiration biopsy specimens from children with tuberculous lymphadenitis .Methods Fine-needle aspiration biopsy was performed on 269 children with tuberculous lymphadenitis diagnosed by Shanghai Public Health Clinical Center from January 2011 to September 2013 .The needle aspiration biopsy specimens were processed for acid-fast bacillus (AFB) smear test, mycobacterial culture and Mycobacterium identification ( p-nitrobenzoic acid inhibition test ) .Results Cytological diagnosis of tuberculous lymphade-nitis was made for 269 patients.The positive results by AFB smear test were detected in 63.19% of 269 specimens (n=170) and 40.15%(n=108) specimens were positive in mycobacterial culture .The differ-ence between the two tests were significant (P<0.01).The positive rate of Mycobacterium detected by using BACTEC MGIT 960 automated system and L?wenstein-Jensen culture method were 38 .66% ( n=104 ) and 28.99%(n=78), respectively, showing the significant difference between two tests (P<0.05).AFB smear test in combination with mycobacterial culture could precisely diagnose 70.63% of tuberculous lym-phadenitis in children.Of the 108 clinical isolates, 105 strains (97.2%) were Mycobacterium tuberculosis complex and the rest were non-tuberculous Mycobacterium strains (2.8%).Conclusion The positive rate by AFB smear test was significantly increased in fine needle aspiration biopsy specimens after a series of treatments including sample digestion , centrifugation and precipitation , but the positive rate of mycobacterial culture was reduced .Diagnostic accuracy could be significantly improved by using BACTEC MGIT 960 sys-tem.Mycobacterium tuberculosis complex was the predominant pathogenic bacterium in children with tubercu-lous lymphadenitis .
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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.
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Objective To establish the rapid pathogen identification method for HIV and Mycobac-terium tuberculosis (Mtb)co-infection and the assay for the drug susceptibility. Methods Geneprobe and 16S rDNA sequencing were used to differentiate mycobacterium species and modified microscopic observation drug susceptibility (MODS) was used for the drug susceptibility test. The above assays were compared with acid-fast smear, L-J culture and proportional drug susceptibility tests. Results (1) Thirty-four mycobacte-rial isolates were obtained from 112 samples collected from 68 HIV patients. Among these isolates, the strain species were determined by Geneprobe and 16S rDNA sequencing as the followings: 21 Mtb complex, 10 NTM including 5 M.avium complex, 2 M.gordonae, 2 M.kansasii, 1 M.colombiense, and 3 co-infection. (2) The sensitivity of Mtb to rifampicin, ethambutol, isoniazid and streptomycin were 100%, 100%, 76.2%, 90.5% respectively, while the sensitivity of NTM to rifampicin, ethambutol, isoniazid and strepto-mycin were 40%, 60%, 0%, 30% respectively. There is no significant statistic difference between the two methods, MODS and the reference standard, for the drug susceptibility test. (3) Six to eight weeks are nee-ded for the identification of the species of mycobacteria and the drug susceptibility test by using traditional method. In this study, 5-14 d, 6-15 d and 10-14 d are needed for Geneprobe, 16S rDNA sequencing, and MODS respectively. The time for the testing has been dramatically shortened. Conclusion The identifica-tion of mycobacterial species and the drug susceptibility test using clinical samples could be completed within 15 days by using combined Geneprobe, 16S rDNA sequencing and modified MODS. This combined method can be used for the pathogen identification and drug resistant test in HIV patients who are co-infected by my-cobacteria.