| Objective:This study was to investigate the colony morphology of L-form M.tuberculosis,the isolation rate of L-form bacteria in clinical samples,the drug sensitivity to commonly used anti-tuberculosis drugs and its association with drug-resistant retreatment of tuberculosis patients.Study and analysis of the association between M.tuberculosis L-form and re-treatment of tuberculosis patients.Methods:1.M.tuberculosis standard strain was inoculated into MGIT 960 mycobacterial liquid medium which containing 0.1 ?g/mL,0.2 ?g/mL rifampicin,0.04 ?g/mL,0.08 ?g/mL isoniazid,2.5 ?g/mL,5.0 ?g/mL,The MTB-L induction culture was carried out on the M.tuberculosis standard strain using a BACTEC MGIT 960 mycobacteria-specific incubator.After the instrument reported positive or the instrument is negative for 40 to 45 days,the culture solution is aspirated with a disposable syringe,the formation of L-form of Mycobacterium tuberculosis was observed by acid-fast staining.The DNA of the induced strain was extracted,and the DNA of the tuberculosis was amplified to confirm the nature of the strain and eliminate the interference of the bacteria.The MTB-L strain confirmed by DNA amplification was transferred to L-form solid medium.2.Configure 92-3 TB-L liquid medium and configure the modified 92-3 TB-L liquid medium for this study on the basis of 92-3 TB-L liquid medium.In order to understand the culture performance of L-form bacteria between the two culture media,20 specimens of smear-positive pulmonary tuberculosis patients were randomly selected for clinical examination.After pretreatment,the above two media and MGIT 960 mycobacterial liquid medium were simultaneously inoculated,and the experiment was repeated 3 times.To verify the results of repeated experiments,the samples were expanded to 50 cases for inoculation.After the instrument is reported to be positive or the instrument is negative after 40 to 45 days of culture,the MGIT 960 mycobacterial liquid culture tube cultured in the instrument is taken out.92-3 TB-L and Self-developed modified 92-3 TB-L liquid medium inoculated in the incubator with the same specimen were also taken out.The liquids in the three culture tubes were simultaneously aspirated to observe the colony formation by acid-fast staining.At the same time,L-form bacteria grows slowly.If MGIT 960 has acid-fast staining positive bacteria,and 92-3 TB-L and Self-developed modified 92-3 TB-L liquid medium do not have L-form bacteria,it needs continue grow to 40~45 days.3.A total of 222 sputum and lavage fluid specimens were collected from smear-positive pulmonary tuberculosis patients who were treated at the tuberculosis clinic from September 2017 to December 2017.The culture was carried out using a MGIT 960 culture tube and the Self-developed modified 92-3 TB-L liquid medium After the instrument alarmed,the strain in the MGIT 960 mycobacterial culture tube was identified by M.tuberculosis MPT64.At the same time,remove the same specimen inoculated in Self-developed modified 92-3 TB-L liquid medium,the formation of bacteria was observed by acid-fast staining and Gram staining.If there is no bacterial growth under the staining microscope,it is necessary to continue culture for 40 to 45 days.The strain with the L-form bacterial characteristics under the microscope was subjected to DNA extraction,and M.tuberculosis DNA amplification was carried out to clarify the nature of the strain and eliminate the interference of the contaminating bacteria.After DNA amplification,the strain confirmed to be M.tuberculosis L-form was transferred to a new Self-developed modified 92-3 TB-L liquid medium and L-form solid medium for subculture to remove the strain that could not be subcultured.The L-J medium was prepared,and the immunized MTB-L strain was subjected to the ancestors to observe the ability of the MTB-L form ancestor to be the parental Mycobacterium tuberculosis.4.The isolated MTB strain and the MTB-L strain were selected.Check the patient's medical history and record the initial retreatment,drug resistance and treatment of the patient.Drug sensitivity test of MTB strain by absolute concentration method.Drug sensitivity test of MTB-L strain by micro MIC method..The gene chip detection method was used to detect the mutation site of the ropB gene related to RFP resistance and the Kat G and inh A genes related to INH resistance.5.This study has been reviewed by the Hospital Medical Ethics Committee.The data was collated with Excel 2010 software,and statistical analysis was performed using SPSS 18.0.Results:1.The rifampicin of 0.1 ?g/mL can induce the M.tuberculosis standard strain into an L-form strain.0.2 ?g/mL rifampicin and other drugs failed to induce M.tuberculosis standard strain successfully.The acid-fast staining of induced strains showed negative and positive coexistence characteristics."Fried egg" colonies were present in the solid medium.2.Comparison of culture performance of 92-3 TB-L liquid medium and Self-developed modified 92-3 TB-L liquid medium.1.Repeat the experiment results to show:The growth rate of self-developed 92-3 TB-L liquid culture medium was 36.8% higher than that of 92-3 TB-L,and the growth rate of mixed bacteria was 61.54% lower than that of 92-3 TB-L.2.Expanded sample culture results show:Among the 50 specimens collected from clinical specimens,BACTEC MGIT 960 Mycobacterium culture apparatus,38 cases were positive,and the positive rate was 76.0%.There were 4 strains in 92-3 TB-L liquid medium,the positive rate was 8.0%,14 other bacteria grew,and the contamination rate was 28.0%.In the Self-developed modified 92-3 TB-L liquid medium,7 strains were grown,the positive rate was 14.0%,4 other bacteria were grown,and the contamination rate was 8.0%.The difference between the three culture methods was statistically significant(?2=38.21,P<0.05).3.152 cases of mycobacterial strains were obtained by BACTEC MGIT 960 mycobacterial culture apparatus.After the confirmation of MP64,there were 147 cases of bacterial type and 5 cases of non-tuberculous mycobacteria.The positive rates were 66.2%(147/222)and 2.3%(5/222).52 strains were obtained by initial culture of Self-developed modified 92-3 TB-L liquid medium.After subculture,acid-fast staining,Gram staining,and tuberculosis DNA amplification,39 strains were confirmed to be MTB-L,and the positive rate was 17.6%(39/222).4.There are three different types of colonies on the solid medium: "granular-like","fried egg-like","filamentous" colonies.The acid-resistant dyeing of MTB-L is characterized by blue and purple-red coexistence and uneven coloration.MTB-L Gram staining showed uneven staining,partial fragmentation loss and also a bead-like Gram-negative and positive staining pattern.Of the 39 cases of MTB-L strains,29 cases could be returned to the parental Mycobacterium tuberculosis,and 10 cases could not be returned to the ancestors.5.There were 222 clinical specimens.A total of 143 patients with complete medical records,clear diagnosis,and complete drug sensitivity data were included.The positive rate of MTB-L in patients with retreatment was 23.9%(17/71),which was greater than 15.0%(22/146)of newly diagnosed patients.However,there was no significant difference in the positive rate of MTB-L between the two groups(?2=2.55,P=0.081).Among the retreated patients,the resistance rate to commonly used anti-tuberculosis drugs was 44.4%(20/45),which was higher than that of newly diagnosed patients(22.4%(22/98)).The difference in drug resistance rates between the two groups was statistically significant(?2=29.81,P=0.000).The proportion of retreated patients in the MTB-L positive cases was 43.6%(17/39),which was higher than that in the MTB-L negative cases(30.3%(54/178)).There was no significant difference in the proportion of patients treated(?2=2.55,P=0.081).The proportion of drug-resistant patients with MTB-L was 37.5%(12/32),which was higher than that of patients without MTB-L(27.0%(30/111),but There was no significant difference in the proportion of drug-resistant cases between the two patients.(?2=1.31,P=0.177).The retreatment rate of patients with MTB-L and MTB bacterial resistance was 66.67%(8/12).The retreatment rate of patients who did not isolate MTB-L and had MTB non-resistance was 24.7%.The retreatment rate of patients who did not isolate MTB-L and had MTB resistance was 40.0%.The retreatment rate of patients with MTB-L and MTB non-resistance was 25.0%.6.MTB-L had a higher resistance rate to isoniazid and rifampicin,and no mutations were detected in drug-resistant gene loci.Conclusion:1.The clinically isolated MTB-L and the induced MTB-L strain have consistency in growth and staining morphology.L-form culture can be modified by self-developed 92-3 TB-L liquid medium.L-form culture can be modified by self-developed 92-3 TB-L liquid medium.2.Tuberculosis patients have a mixed infection of MTB-L and MTB.For patients with smear-positive pulmonary tuberculosis who are sent for multiple tests and cultures are negative,the culture of L-form bacteria should be considered.3.During the treatment of tuberculosis patients,clinicians should pay attention to the formation of L-forms.At this time,the combination of sensitive antibacterial drugs for MTB-L form should be used.For patients with newly diagnosed and drug-resistant tuberculosis,MTB-L should be tested.When the MTB-L culture is positive,the patient is more likely to have a recurrence. |
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