Molecular characterization of the rpoB gene mutations of Mycobacterium tuberculosis isolated from China

Objective: To analyze characterization of the rpoBgene mutations of Mycobacterium tuber- culosis isolated from China and to explore the association of specific mutations conferring rifampicin (RIF) resistance with Beijing genotype strains. Methods: Genotypic analysis of 3479M. tuberculosis isolatesincluding 402 RIF-resistantand 3077 RIF-susceptible isolated from the na- tional drug-resistant tuberculosis baseline survey was performed. Results: DNA sequencing analysis of the 81-bp RIF resistance determining region (RRDR) of the ropB gene revealed that 98.01% of RIF-resistant strains showedrpoBgene mutation, isolates with mutations at codon rpoB531, rpoB 526 and rpoB 516 were the most frequently. Analysis of the rpoB gene of 3077 RIF-susceptible strains revealed that 98.96% of the strains had no mutation. The distribution of mutation frequency at differentcritical codons in different regions of China was statistically significant (p = 0.001). There was no significant difference in the occurrence of mutations at critical codons between the rifampicin-resistant Bei-jing and non-Beijing isolates.Conclusion: About 98% of RIF-resistant strains isolated from China carry mutations in RRDR ofrpoB gene.Mutation profiles in RIF-resistantM. tuberculosis clinical isolates are variable depending on the different geographical regionsof China. The results provide valuable information in adopting new molecular methods for diagnosis of TB in China.

Characteristics of compensatory mutations in the rpoC gene and their association with compensated transmission of Mycobacterium tuberculosis

The aim of this study was to characterize rpoC gene mutations in Mycobacterium tuberculosis(MTB)and investigate the factors associated with rpoC mutations and the relation between rpoC mutations and tuberculosis(TB)transmission.A total of 245 MTB clinical isolates from patients with TB in six provinces and two municipalities in China were characterized based on gene mutations through DNA sequencing of rpoC and rpoB genes,phenotyping via standard drug susceptibility testing,and genotypic profiling by mycobacterial interspersed repetitive unit-variable number tandem repeat(MIRU-VNTR)typing.Approximately 36.4%of the rifampinresistant isolates harbored nonsynonymous mutations in the rpoC gene.Twenty-nine nonsynonymous single mutations and three double mutations were identified.The rpoC mutations at locus 483(11.3%)were predominant,and the mutations at V483G,W484G,I491V,L516P,L566R,N698K,and A788E accounted for 54.5%of the total detected mutations.Fifteen new mutations in the rpoC gene were identified.Rifampin resistance and rpoB mutations at locus 531 were significantly associated with rpoC mutations.MIRU-VNTR genotype results indicated that 18.4%of the studied isolates were clustered,and the rpoC mutations were not significantly associated with MIRU-VNTR clusters.A large proportion of rpoC mutation was observed in the rifampicin-resistant MTB isolates.However,the findings of this study do not support the association of rpoC mutation with compensated transmissibility.

National Proficiency Testing of Molecular Diagnostics forTuberculosis and Drug Resistance Detection-China,2014-2019

Summary What is already known about this topic?Proficiency testing(PT)is a key component of qualityassurance and is essential in ensuring accuratelaboratory diagnosis of tuberculosis(TB)and drug-resistant TB.The National Tuberculosis ReferenceLaboratory(NTRL)developed a novel PT panel to testlaboratories’proficiency for molecular TB diagnosticassays throughout the TB laboratory network.A totalof 6 PT rounds for molecular diagnostics wereconducted by NTRL from 2014 to 2019.

Characterization of isoniazid resistance and genetic mutations in isoniazid-resistant and rifampicin-susceptible Mycobacterium tuberculosis in China

Background Patients with tuberculosis resistant to isoniazid but susceptible to rifampicin(Hr-Rs TB)remain a neglected demographic,despite a high disease burden and poor outcomes of these patients.The aim of this study was to investigate the characteristics of isoniazid-resistance-related mutations in Mycobacterium tuberculosis and resistance rates to drugs included in WHO-recommended regimens for Hr-Rs patients.Methods Mycobacterium tuberculosis isolates(n=4922)obtained from national tuberculosis drug-resistance surveillance were subjected to whole-genome sequencing to identify Hr-Rs strains.The minimal inhibitory concentrations(MICs)were established for the Hr-Rs strains to determine the isoniazid resistance levels.We also identified drug-resistance-associated mutations for five drugs(fluoroquinolones,ethambutol,pyrazinamide,streptomycin,and amikacin)in the Hr-Rs strains.Results Of the 4922 strains,384(7.8%)were Hr-Rs.The subculture of seven strains failed,so 377(98.2%)strains underwent phenotypic MIC testing.Among the 384 genotypic Hr-Rs strains,242(63.0%)contained the katG Ser315Thr substitution;115(29.9%)contained the-15C>T in the promoter region of the fabG1 gene;and 16(4.2%)contained Ser315Asn in the katG gene.Of the 239 strains with the Ser315Thr substitution,229(95.8%)had MIC≥2µg/mL,and of the 114 strains with the-15C>T mutation,103(90.4%)had 0.25µg/mL≤MIC≤1µg/mL.The genotypic resistance rates were 0.8%(3/384)for pyrazinamide,2.3%(9/384)for ethambutol and fluoroquinolones;39.6%(152/384)of the strains were resistant to streptomycin,but only 0.5%(2/384)of the strains were resistant to amikacin.Conclusion Ser315Thr in katG was the predominant mutation conferring the Hr-Rs phenotype,followed by the fabG1-15C>T mutation.The combination of rifampicin,pyrazinamide,ethambutol,and levofloxacin should be effective in the treatment of patients with Hr-Rs tuberculosis because the resistance rates for these drugs in China are low.