| Objective: China is one of the three countries with the most severe multidrug resistant tuberculosis（MDR-TB）in the world.The aim of our study was to analyze the clinical characteristics,imaging features and risk factors of children with drug-resistant tuberculosis（DR-TB）and MDR-TB.Methods: All Mycobacterium tuberculosis（Mtb）strains were isolated from patients during routine practice of Children’s Hospital of Chongqing Medical University in China,from July 2015 to November 2020.The isoniazid,rifampicin,ethambutol,rifapentine,amikacin,prothionamide,levofloxacin and moxifloxacin resistance of Mtb strains were detected by ratio method drug susceptibility test（DST）,and the genotypes of the Mtb strains were identified by whole-genome sequencing（WGS）.According to the results of the ratio method DST and WGS,all children were divided into different groups.According to the definition of severe tuberculosis in children,all 208 children were divided into severe group and non-severe group.The population characteristics,medical data,and treatment outcome of children in each group were analyzed retrospectively.Results: A total of 208 children were included in the study,among whom 201 children were divided into the drug-resistant groups,including13 cases（13/201,6.5%）in the MDR group,30 cases（30/201,14.9%）in the DR group and 171 cases（171/201,85.1%）in the drug susceptible（DS）group.Among 208 children,146 cases（146/208,70.2%）were in the severe group,62（62/208,29.8%）were in the non-severe group,154（154/208,74.0%）were in the lineage 2 group,and 54（54/208,26.0%）were in the lineage 4 group.There was no significant difference in the distribution of gender,age,nationality,residence,residence,admission,history of tuberculosis exposure,duration of symptoms before effective treatment,anti-tuberculosis treatment,and treatment outcome in the MDR,DR and DS group（P>0.05）.The Bacille Calmette-Guérin vaccine（BCG）vaccination rate in the MDR group was lower than that in the DS group,with significant difference（P<0.05）.The rate of severe TB was 100.0%（13/13）in the MDR group,which was significantly higher than that in the DS group（115/171,67.3%,P<0.05）.In the MDR group,92.3%（12/13）of patients were diagnosed with pulmonary tuberculosis complicated with extrapulmonary tuberculosis,and 30.8% of patients with adverse treatment outcome,which were higher than that in the DS group（52.6% and 6.4%,P<0.05）.There were significant differences of hospitalization times,first hospitalization days,diagnosis,treatment and outcome between the severe and non-severe group（P<0.05）.According to the results of binary logistic regression,the risk of MDR-TB in children without BCG vaccination was4.39 times higher than that with BCG vaccination（OR=0.228）.MDR-TB was a risk factor for pulmonary tuberculosis complicated with extrapulmonary tuberculosis（OR=13.386）.Conclusion: Compared with DS tuberculosis,children with MDR-TB had a lower BCG vaccination rate.The BCG vaccination was a protective factor for MDR-TB.MDR-TB might not only induce pulmonary infection,but tends to spread to extrapulmonary organs in children.There were more severe cases in children with MDR-TB than that with DS tuberculosis.The proportion of failure treatment or death was higher in children with severe tuberculosis and MDR-TB than those with non-severe tuberculosis and DS-TB.Objective:The aim of this study was to evaluate the validation of WGS in the detection of drug susceptibility of Mtb in children.Methods:All of the 208 Mtb strains were successfully recovered,then were tested for the resistance of isoniazid,rifampin,ethambutol,rifapentine,amikacin,prothionamide,levofloxacin and moxifloxacin using ratio method DST and WGS.The ratio method DST was used as the gold standard,and the consistency test analysis was used to evaluate the consistency of WGS and ratio method DST.Results:According to the result of ratio method DST,82.21%（171/208）of Mtb strains were susceptible to all eight drugs,17.79%（37/208）of strains were resistance to at least one drug,which was consistent with the result of WGS.Using the ratio method DST as the gold standard,the kappa result of WGS to predict isoniazid,rifampin,rifapentine,amikacin,prothionamide,levofloxacin and moxifloxacin were0.84,0.89,0.59,0.86,0.89,0.82,0.88,and 1.00,respectively.The sensitivity,specificity,positive predictive value and negative predictive value for WGS to predict drug resistance of Mtb were 84.0%,98.4%,87.5% and 97.8% for isoniazid,100.0%,98.5%,81.3% and 100.0% for rifampin,57.1%,97.9%,66.7% and 96.9% for ethambutol,87.5%,99.0%,87.5% and 99.0% for rifapentine,100.0%,99.5%,80.0% and 100.0% for prothionamide,100.0%,98.5%,70.0% and 100.0% for levofloxacin,100.0%,99.0%,80.0% and 100.0% for moxifloxacin,100.0%,100.0%,100.0% and 100.0% for amikacin,respectively.Conclusion:The result of WGS in predicting isoniazid,rifampicin,rifapentine,prothionamide,levofloxacin,moxifloxacin,and amikacin susceptibility of Mtb strains in children,was in strong agreement with the result of ratio method DST.WGS could be used for clinical diagnosis in children.However,the result of WGS in predicting the susceptibility of ethambutol was not consistent with the ratio method DST.It is suggested to use WGS and ratio method DST jointly to evaluate the susceptibility of ethambutol.Objective:The aim of this study was to identify mutations in genes related to drug susceptibility of Mtb strains,which were isolated from children with TB,find new mutations and verify their functions.Methods:（1）The result of drug susceptibility of 208 Mtb strains to isoniazid,rifampicin,ethambutol,prothionamide,rifapentine,levofloxacin,moxifloxacin and amikacin were detected by the ratio DST.WGS was used to identify the mutations in genes related to the susceptibility of eight anti-tuberculosis drugs.（2）The target gene of Rv3795Ala356 Val was amplified by polymerase chain reaction,and then was integrated into p MV261 vector by seamless cloning method.The plasmid was extracted and sequenced.The positive plasmids were transferred into the wild-type strain of Mycobacterium smegmatis by electro transformation,and the overexpression strains were obtained.The minimum inhibitory concentration（MIC）of ethambutol were identified by 96 liquid plates.Results:（1）Among the 25 Mtb strains identified as isoniazid resistant by ratio method DST,21 strains had three mutations,including kat G Ser315Thr（19/25,76.0%）,fab G1-15C>T（2/25,8.0%）and inh A Ile194Thr（1/25,4.0%）.There was significant difference in the distribution of kat G Ser315 Thr and fab G1-15C>T mutations between isoniazid resistant and susceptible strains（P<0.05）.There was significant difference in the distribution of eth A 140140del、eth A 938939ins T、fab G1-15C>T and inh A Ile194 Thr mutations between prothionamide resistant and susceptible strains identified by ratio method DST（P<0.05）.The latter two mutations were related to cross-resistance of isoniazid and prothionamide.The distribution of mutations at codons 445 and 435 in rpo B gene between rifampicin resistant and susceptible strains was statistical different（P<0.05）.In ethambutol resistant strains,the most commonly mutated codons were306,accounting for 35.7%（5/14）,followed by 406（14.3%,2/14）.The distribution of mutations at codons 306 and 406 in emb B gene beween ethambutol resistant and susceptible strains was statistical different（P<0.05）.There was no significant difference in the distribution of Ala356 Val mutation between the ethambutol resistant and susceptible strains（P>0.05）.（2）The strains of Mycobacterium smegmatis overexpressing Rv3795 Ala356 Val were successfully constructed.The MICs of Mycobacterium smegmatis overexpressing Rv3795 Ala356 Val strains and wild-type Mycobacterium smegmatis strains to ethambutol were the same,with the MIC value as 5μg/m L.Conclusion:The types and frequency distribution of mutations in genes related with drug resistance of Mtb strains,which were isolated from children,were comparable to previous studies,and no new mutations were identified.The mutation rate of kat G Ser315 Thr in isoniazid resistant Mtb strains was the highest,so it is not recommended to treat isoniazid resistant TB using high-level isoniazid.It is suggested that to clarify the DST result of prothionamide,for the treatment of MDR-TB.The Ala356 Val mutation in emb B gene could not enhance the MIC value of ethambutol resistance of Mycobacterium smegmatis,which might not be contribute to the emergence of ethambutol-resistant strains. |
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