| Objective:Species identification and drug susceptibility tests were carried out on acid-fast bacilli collected from hospitals in Changzhou city, in order to understand the species distribution and their drug resistances and to analyze the reasons to the resistances accordingly. Results would be provided as references for the diagnosis and rational medication of tuberculosis and nontuberculous mycobacteria(NTM) pulmonary diseases in this region.Methods:1. Totally 400 copies of acid-fast bacilli for species identification and drug resistance study were collected from Changzhou special hospitals in April of 2011 to March of 2013, including 300 copies of sputum, 70 of pleural effusion and 30 of bronchoalveolar lavage fluids.2. After being dealt through Na OH-Na Cl sample processing method, the 400 specimens were undergone identifications of bacterial type and drug resistance as follows:(1)bacterial type identification: the specimens were firstly isolated by Bac T/Alert 3D system and then incubated separately, followed by acid-fast staining and Gram staining. Next, the acid-fast positive specimens were measured by Gen-probe test kit with Accu Probe probe to detect flora of NTM and MTB.(2)resistance test: the positive specimens of Mycobacterium strains were detected by absolute concentration method on the following six anti-TB drugs: INH(isoniazid), RFP(rifampicin), EMB(ethambutol), SM(streptomycin), LFX(levofloxacin) and AMK(amikacin)(Low and high concentrations were set for each drug).3. Gene chip detection: gene chip tests were applied to all specimens to detect mutation status of 6 genes on site rpo B of RFP and 2 promoters on genes Kat G and inh A of INH, one for each site. Specimens were applied with nucleic acid extraction, followed by PCR amplification and hybridization to separate and identify acid-fast bacterial types. Lux Scan10k-B microarray chip scanner was used with the corresponding identification system to scan, read and record test results.Results:1. Results of bacterial tests: Flora identification: out of 400 suspicious specimens, 191 copies were found positive in acid-fast bacilli strains tests, including 175 MTB strains(91.62%) and 16 NTM strains(8.38%), and 209 were negative.2. Resistance Results:(1) comparison of MTB and NTM: Among 175 MTB strains, 63.43% cases were susceptive to all 6 antituberculotic drugs. Moreover, the ratios of bacterial strains to each drug resistance were found as 16.57% for INH, 12.00% for RFP, 9.14% for EMB, 16.00% for the SM, 15.43% for LFX and 9.14% for AMK. Regarding NTM strains, 23.08% cases were susceptive to all 6 antituberculotic drugs. And there were only 13 out of 16 cases showed drug resistance, including 76.92% of INH resistance, 38.46% of RFP resistance, 53.85% of EMB resistance, 61.54% of SM resistance, 61.54% of LFX resistance and 46.15% of AMK resistance.(2)Susceptibility differences between MTB and NTM to drugs with varied concentrations. For MTB strains, the highest resistance rate to drug of low concentration was occurred to LFX, showing 17.42%, following by EMB of 7.30%, INH of 7.30%,AMK of 6.18%,SM of 5.62% and RFP of 5.06%.Meanwhile, general resistances of all investigated drugs were indicated in the 13 NTM strains, with highest resistance rate of 50.00% for low concentration of EMB, and in turns of 41.67% of LFX, 33.33% of SM,25.00% of INH,25.00% of AMK and 16.67% of RFP.3. Gene microarray:(1)Strain identification:the positive rate of 191 positive acid-fast bacilli specimens was 100% by gene chips. And 5 cases of MTB, 2 cases of NTM were additionally detected in 209 negative specimens.(2)Resistance gene detection: The consistent rates between gene chip and conventional resistance analysis-was 90.1%(172/191) for RFP-resistance, 79.3%(23/29) for RFP-susceptibility, 98.2%(159/162) for specificity, 88.5%(23/26) for predictive positive value, and 96.4%(159/165) for predictive negative value. In respective to INH, the consistent rates between gene chip and conventional analysis were 93.7%(179/191) for resistance, 87.1%(34/29) for susceptibility, 95.4%(145/152) for specificity, 82.9%(34/41) for predictive positive value and 96.0%(145/151) for predictive negative value. Besides, the consistent rates of multi-drug resistance tests between gene and conventional ways were 96.7%(173/191) for resistance, 86.7%(52/60) for sensitivity, 92.4%( 121/131) for specificity, 83.9%(52/62) for predictive positive value and 93.8%(121/129) for negative predictive value.Conclusion:1. In Changzhou region, high overall resistance rates of MTB, isolation rate is higher than the national level;pecimens NTM isolation rate below the national level.2. Microarray method showed relatively high consistency compared with conventional way for identification of acid-fast bacilli strain types and their resistance studies. Moreover, the gene method was fast, efficient and of high specificity, which is able to be recommended in clinical practice.3. Among different specimens, bronchoalveolar lavage fluid showed highest ratio of acid fast bacilli and multi-drug resistancy, followed by sputum and chest water. |
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