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Despite antibiotic treatment and Bacille Calmette-Guerin (BCG) vaccination, Mycobacterium tuberculosis remains a major public health burden in most developing countries. Therefore, developing an improved vaccine is high priority. In this study, we cloned the genes of the immunodominant antigen of M. tuberculosis viz. its 38-kDa antigen (Pst homolog) (Rv0934, PstS1), and its T cell epitopes (amino acid [aa]169–405 and [aa]802–1119), which we termed PstS1p. Prokaryotic expression showed that the two recombinant proteins were mainly in the form of inclusion bodies. We also evaluated the immunity and immunogenicity of PstS1 and PstS1p. Both PstS1 and its T cell epitopes elicited significantly higher antigen-specific immunoglobulin G (IgG) antibodies in mouse serum, indicating that they enhanced antibody response. They also elicited the T helper 1 (Th1)-type response and promoted CD4+ T cell proliferation. Compared to PstS1, PstS1p promoted stronger cell-mediated immune response. These data indicate that PstS1p is highly immunogenic in mice, and may be a promising candidate vaccine for controlling tuberculosis.

Ma-chao Li,1,* Jie Lu,2,* Yao Lu,1,3,* Tong-yang Xiao,4,* Hai-can Liu,1 Shi-qiang Lin,5 Da Xu,1 Gui-lian Li,1 Xiu-qin Zhao,1 Zhi-guang Liu,1 Li-li Zhao,1 Kang-lin Wan1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, People’s Republic of China; 3School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, People’s Republic of China; 4Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, People’s Republic of China; 5Department of Bioinformatics, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-li Zhao; Kang-lin WanNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing, 102206, People’s Republic of ChinaTel/Fax +86 10 58900779Email zhaolili@icdc.cn; wankanglin@icdc.cnObjective: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.Methods: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.Results: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.Conclusion: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.Keywords: Mycobacterium tuberculosis, rifampin resistance, structure, mutation

Aiketaguli Anwaierjiang,1,* Quan Wang,2,* Haican Liu,3,* Chunjie Yin,1 Miao Xu,2 Machao Li,3 Mengwen Liu,1 Yan Liu,2 Xiuqin Zhao,3 Jinbao Liu,1 Guilian Li,3 Xiaokaiti Mijiti,2 Kanglin Wan3 1College of Public Health, Xinjiang Medical University, Wulumuqi, 830011, People’s Republic of China; 2The Eighth Affiliated Hospital of Xinjiang Medical University, Wulumuqi, 830001, People’s Republic of China; 3State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinbao Liu; Guilian Li Email 283474978@qq.com; liguilian@icdc.cnObjective: Drug-resistant tuberculosis is a major public health problem, especially in the southern region of Xinjiang, China; however, there is little information regarding drug resistance profiles and mechanism of Mycobacterium tuberculosis in this area. The aim of this study was to determine the prevalence and molecular characteristics of M. tuberculosis resistant to four anti-tuberculosis drugs from this area.Methods: Three hundred and forty-six isolates from the southern region of Xinjiang, China were included and used to perform phenotypic drug susceptibility testing and whole genome sequencing (WGS). Mutations in seven loci associated with drug resistance, including rpoB for rifampicin (RMP), katG, inhA promoter and oxyR-ahpC for isoniazid (INH), rrs 530 and 912 loops and rpsL for streptomycin (STR), and embB for ethambutol (EMB), were characterized.Results: Among 346 isolates, 106, 60, 70 and 29 were resistant to INH, RMP, STR and EMB, respectively; 132 were resistant to at least one of the four anti-tuberculosis drugs and 51 were multi-drug resistant (MDR). Beijing genotype and retreated patients showed a significantly increased risk for developing MDR tuberculosis. Compared with the phenotypic data, the sensitivity and specificity for WGS to predict resistance were 96.7% and 98.6% for RMP, 75.5% and 97.1% for INH, 68.6% and 99.6% for STR, 93.1% and 93.7% for EMB, respectively. The most common mutations conferring RMP, INH, STR and EMB resistance were Ser450Leu (51.7%) in rpoB, Ser315Thr (44.3%) in katG, Lys43Arg (35.7%) in rpsL and Met306Val (24.1%) in embB.Conclusion: This study provides the first information on the prevalence and molecular characters of drug resistant M. tuberculosis in the southern region of Xinjiang, China, which will be helpful for choosing early detection methods for drug resistance (ig, molecular methods) and subsequently initiation of proper therapy of tuberculosis in this area.Keywords: Mycobacterium tuberculosis, whole-genome sequencing, resistance, prevalence, mutation, isoniazid, rifampicin, streptomycin, ethambutol

Background Reliable and timely determination of second-line drug resistance is essential for early initiation effective anti-tubercular treatment among multi-drug resistant (MDR) patients and blocking the spread of MDR and extensively drug-resistant tuberculosis. Molecular methods have the potency to provide accurate and rapid drug susceptibility results. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of fluoroquinolones (FQs), kanamycin (KN), amikacin (AMK), capreomycin (CPM) and second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis. Methods We established and evaluated the accuracy of the RDBH assay by comparing to the phenotypic drug susceptibility testing (DST) and sequencing in 170 M. tuberculosis, of which 94 and 27 were respectively resistant to ofloxacin (OFX) and SLIDs. Results The results show that, compared to phenotypic DST, the sensitivity and specificity of the RDBH assay for resistance detection were 63.8% and 100.0% for OFX, 60.0% and 100.0% for KN, 61.5% and 98.1% for AMK, 50.0% and 99.3% for CPM, and 55.6% and 100% for SLIDs, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 95.2% and 100.0% for OFX, 93.8% and 100.0% for SLIDs or KN (both based on mutations in rrs 1400 region and eis promoter), and 91.6% and 100.0% for AMK or CPM (both based on mutations in rrs 1400 region), respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples. Conclusion Our data suggested that compared to sequencing, the RDBH assay could serve as a rapid and reliable method for testing the resistance of M. tuberculosis against OFX and SLIDs, enabling early administration of appropriate treatment regimens among MDR tuberculosis patients.

Objectives Pasiniazid is a chemical complex of isoniazid (INH) and para-aminosalicylic acid (PAS). The aim of this study was to explore the cross-resistance of INH, PAS and pasiniazid against INH-resistant Mycobacterium tuberculosis isolates in China. Methods A Microplate alamarBlue® Assay was performed to determine the minimum inhibitory concentrations (MICs) of INH, PAS and pasiniazid against 109 INH-resistant M. tuberculosis isolates. A statistical analysis of the relationship between different genotypes, gene mutations, and INH, PAS or pasiniazid susceptibility was then performed. Results Among the 109 INH-resistant isolates, 13 (11.9%) and 21 (19.3%) showed resistance to PAS and pasiniazid, respectively. Among the 13 PAS-resistant M. tuberculosis isolates, 11 remained susceptible to pasiniazid. Of 63 INH-resistant isolates harbouring mutations in katG, the inhA promoter or the oxyR–ahpC intergenic region, 52 remained susceptible to pasiniazid. Moreover, 11 of 13 pasiniazid-resistant isolates carried mutations in katG, the inhA promoter or the oxyR–ahpC intergenic region. Conclusion Taken together, these results demonstrate that PAS resistance and mutations in thekatG gene, inhA promoter or oxyR–ahpC intergenic region in INH-resistant M. tuberculosis have little effect on pasiniazid susceptibility.

Objectives To explore the prevalence, risk and genetic characteristics of drug-resistant tuberculosis (TB) from a tertiary care TB hospital in China. Patients and methods We carried out a retrospective study including isolates from 189 patients with pulmonary TB at Fuzhou Pulmonary Hospital. All isolates from these patients were subjected to drug susceptibility testing and genotyping. For drug-resistant isolates, DNA sequencing was used to investigate mutations in 12 loci, including katG, inhA, oxyR-ahpC, rpoB, rpsL, rrs1 (nucleotides 388-1084 of rrs), embB, tlyA, eis, rrs2 (nucleotides 1158-1674 of rrs), gyrA and gyrB. Results Among 189 isolates, 28.6% were resistant to at least one of the seven anti-TB drugs, including isoniazid (INH), rifampin (RIF), streptomycin (STR), ethambutol (EMB), capreomycin (CAP), kanzmycin (KAN) and ofloxacin (OFX). The proportion of multidrug-resistant TB and extensively drug-resistant TB isolates was 9.5% and 1.1%, respectively. Patients in rural areas as well as previously treated patients showed a significantly increased risk of developing drug resistance. In addition, among these isolates, 111 (58.7%) were Beijing genotype strains, 84 (75.7%) of which belonged to modern Beijing sublineage. There was no association between genotype and drug resistance. The most common mutations were katG315, rpoB531 rpsL43, embB306, rrs1401 and gyrA94. Conclusion These findings provided additional information of drug-resistant TB in China. Previously treated patients and patients in rural areas should receive greater attention owing to their higher risk of developing drug resistance.

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

ObjectiveTibet has the highest prevalence of both tuberculosis disease and multidrug-resistant tuberculosis (MDR-TB) in China. The circulated Mycobacterium tuberculosis strains from Tibet were sequenced to investigate the underlying drivers for the high burden of MDR-TB.MethodsUsing whole-genome sequencing data of 576 M. tuberculosis strains isolated from consecutive patients in Tibet, we mapped resistance-conferring mutations onto phylogenetic trees to determine their evolution and spread. The impact of drug resistance on bacterial population growth was assessed with a Bayesian (Skyline Plot) analysis. Multivariable logistic regression was used to identify risk factors for the development of rifampicin resistance.ResultsOf the 576 isolates, 284 (49.3%), 280 (48.6%), and 236 (41.0%) were, respectively, genetically resistant to isoniazid, rifampicin, or both (MDR-TB). Among the isoniazid- and rifampicin-resistant strains, the proportions in phylogenetically-inferred clusters were 77.8% (221/284) and 62.1% (174/280), respectively. Nearly half (47.2%, 134/284) of the isoniazid-resistant strains were in six major clades, which contained between 8 and 58 strains with katG S315T, katG S315N, or fabG1 promoter −15 C>T resistance mutations. These major clades exponentially expanded after emerging with isoniazid resistance and stabilized before evolving into MDR-TB twenty years later. Isoniazid-resistant isolates showed an increased risk of accumulating rifampicin resistance compared to isoniazid-susceptible strains, with an adjusted odds ratio of 3.81 (95% confidence interval 2.47-5.95).ConclusionHistorical expansion of isoniazid-resistant strains and their increased likelihood of acquiring rifampicin resistance both contributed to the high burden of MDR-TB in Tibet, highlighting the need to detect INH-resistant strains promptly and to control their transmission.

Background Since the 1980s, China has undergone significant social change and the incidence of infectious diseases has also changed considerably. Here, we report the epidemiological features and changes in notifiable infectious diseases in China from 1986 to 2016 to explore the factors contributing to the successful control of infectious diseases and the challenges faced in the prevention and control of infectious diseases. Methods The data of notifiable infectious diseases in China from 1986 to 2016 were collected from the monthly analysis report of the National Infectious Disease Surveillance System. Joinpoint regression models were used to examine incidence and mortality trends from 1986 to 2016. IBM SPSS Statistics version 22.0, Excel 2010 and R x64 3.5.2 were used for data analysis. Results A total of 132 858 005 cases of notifiable infectious diseases were reported over these 31 years, with an average yearly incidence of 342.14/100 000. There were 284 694 deaths with an average yearly mortality rate of 0.73/100 000. The overall incidence and overall mortality of notifiable infectious diseases both showed a "U" distribution (ie, a decrease, stable, an increase, stable again). The top five diseases in terms of incidence were hand, foot and mouth disease, viral hepatitis, tuberculosis, other infectious causes of diarrhea and dysentery, accounting for 78.0% of all reported cases. The top five causes of death were HIV/AIDS, rabies, tuberculosis, viral hepatitis and epidemic encephalitis B, which accounted for 76.07% of all mortalities. The diseases with the top five fatality rates were rabies, H5N1, H7N9, HIV/AIDS and plague, with rates of 91.06%, 66.07%, 38.51%, 25.19% and 10.31%, respectively. Conclusions This analysis will benefit the future monitoring of infectious diseases and public health measures in China.

Development of modern genomics provides us an effective method to understand the molecular mechanism of drug resistance and diagnose drug-resistant Mycobacterium tuberculosis. In this study, mutations in 18 genes or intergenic regions acquired by whole-genome sequencing (WGS) of 183 clinical M. tuberculosis strains, including 137 multidrug-resistant and 46 pan-susceptible isolates from China, were identified and used to analyze their associations with resistance of isoniazid, rifampin, ethambutol, and streptomycin. Using the proportional method as the gold standard method, the accuracy values of WGS to predict resistance were calculated. The association between synonymous or lineage definition mutations with different genotypes were also analyzed. The results show that, compared to the phenotypic proportional method, the sensitivity and specificity of WGS for resistance detection were 94.2 and 100.0% for rifampicin (based on mutations in rpoB), 90.5 and 97.8% for isoniazid (katG), 83.0 and 97.8% for streptomycin (rpsL combined with rrs 530 loop and 912 loop), and 90.9 and 65.1% for ethambutol (embB), respectively. WGS data also showed that mutations in the inhA promoter increased only 2.2% sensitivity for INH based on mutations in katG. Synonymous mutation rpoB A1075A was confirmed to be associated with the Beijing genotype. This study confirmed that mutations in rpoB, katG, rrs 530 loop and 912 loop, and rpsL were excellent biomarkers for predicting rifampicin, isoniazid, and streptomycin resistance, respectively, and provided clues in clarifying the drug-resistance mechanism of M. tuberculosis isolates from China.

With the increasing incidence of drug-resistant tuberculosis (DR-TB), determining a rapid and accurate drug susceptibility testing (DST) method to identify ethambutol (EMB) resistance in Mycobacterium tuberculosis has become essential for patient management in China. Herein, we evaluated the correlation between three phenotypic DST methods, namely, proportion method (PM), MGIT 960 system, and microplate alamar Blue assay (MABA), and DNA sequencing of embAB in 118 M. tuberculosis isolates from China. When the results of the phenotypic DST methods were compared with those of DNA sequencing, the overall agreement and kappa values of the PM, MGIT 960 system, and MABA were 81.4% and 0.61, 77.1% and 0.55, and 84.7% and 0.67, respectively. The agreement for EMB resistance between MABA and PM was significantly higher than that between the MGIT 960 system and PM (P = 0.02). Moreover, among the isolates with detectable embAB mutations, 97.2% (70/72 isolates) harbored mutations in embB. The analysis of embB mutations predicted EMB resistance with 81.3% sensitivity, 86.8% specificity, and 83.1% accuracy. Thus, MABA may be a better phenotypic DST method for detecting EMB resistance. DNA sequencing of embB may be useful for the early identification of EMB resistance and the consequent optimization of the treatment regimen.

Background Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China. Methods In this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson’s chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing. Results The results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples. Conclusions Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

Background:Klebsiella pneumoniae is an opportunistic pathogen associated with community-acquired and nosocomial infections. Since 2010, K. pneumoniae testing has been included into an existing diarrhea-syndrome surveillance system for estimating the prevalence of K. pneumoniae in diarrhea-syndrome patients, assessing antibiotic susceptibility, and investigating molecular characteristics of K. pneumoniae. Methods:Klebsiella pneumoniae strains were isolated from stool specimens from diarrhea-syndrome outpatients in Beijing, China. Isolates were tested for antibiotic susceptibility, and phylogenetic relationships were explored though whole genome sequence analysis. Multi-locus sequence type (MLST) alleles were extracted from the whole genome sequence (WGS) data. A maximum likelihood tree was generated by MEGAX. Genomes were annotated by Prokka; core genes were produced by Roary; a maximum likelihood phylogenetic tree was generated using FastTree. Results: Forty-four K. pnuemoniae strains were isolated from 2010 to July 2019; of these 37 were K. pneumoniae and seven were K. variicola. Antibiotic susceptibility testing showed that all 44 strains were sensitive to gentamicin, imipenem, amikacin, meropenem, kanamycin; 97.73% were sensitive to cefoxitin andlavo-ofloxacin; the highest antibiotic resistance rate was 79.55%, which was to ampicillin. We found three extended-spectrum beta-lactamase (ESBL) producing strains; we identified high-virulence ST types, including ST307 and ST65; and we found that ST23 has been the epidemic clone since 2010. MLST and core genome sequence analysis showed two distinct clusters of 44 K. pnuemoniae; 40 alleles were identified in core genome sequence analysis, while 36 alleles were identified in MLST typing. Conclusions: There is an urgent need for epidemiological and molecular studies to understand the dynamics of antibiotic resistance and virulence gene transmission to guide strategies for K. pneumoniae surveillance. WGS analysis provided high discrimination power and reliable and robust data useful for molecular epidemiology.

Mycobacterium tuberculosis is the most successful pathogen with multiple mechanisms to subvert host immune response, resulting in insidious disease. There are few studies on whether the bacteria undergo antigenic variation in response to host immune pressure. Studies on T cell epitopes of M. tuberculosis can help us further understand the mechanism of interaction between the bacteria and host immune system. Here, we selected 180 M. tuberculosis complex in China, amplified 462 experimentally verified human T cell epitopes, sequenced and compared the results to analyze the diversity of those epitopes. It proved that a large majority human T cell epitopes of M. tuberculosis are conserved. However, polymorphisms of T cell epitopes indicated different categories of proteins suffered divergence from host immune pressure. Moreover, Beijing strains are more conservative than non-Beijing strains in T cell epitopes, which might make them easier to transmit than non-Beijing strains.

Investigations on the genetic diversity of Mycobacterium tuberculosis in China have shown that Beijing genotype strains play a dominant role. To study the association between the M. tuberculosis Beijing genotype and the drug-resistance phenotype, 1286 M. tuberculosis clinical isolates together with epidemiological and clinical information of patients were collected from the center for tuberculosis (TB) prevention and control or TB hospitals in Beijing municipality and nine provinces or autonomous regions in China. Drug resistance testing was conducted on all the isolates to the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, and ethambutol). A total of 585 strains were found to be resistant to at least one of the four anti-TB drugs. The Beijing family strains consisted of 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains, whereas the non-Beijing family strains comprised 202 (58.05%) drug-sensitive strains and 146 (41.95%) drug-resistant strains. No significant difference was observed in prevalence (χ2= 2.41, P > 0.05) between the drug-resistant and drugsensitive strains among the Beijing family strains. Analysis of monoresistance, multidrug-resistant TB, and geographic distribution of drug resistance did not find any relationships between the M. tuberculosis Beijing genotype and drug-resistance phenotype in China. Results confirmed that the Beijing genotype, the predominant M. tuberculosis genotype in China, was not associated with drug resistance.

Rapid detection of resistance to the second-line drugs is essential for early initiation of appropriate anti-tubercular treatment regimen among multi-drug tuberculosis (MDR-TB). In this study, we applied a multiplex allele-specific PCR (MAS-PCR) to identify the mutations on codons 90 and 94 of gyrA and nucleotide 1401 of rrs for detecting ofloxacin (OFX) and kanamycin (KAN) resistance in 139 MDR-TB isolates from China. Using the traditional phenotypic method as the reference, MAS-PCR detected resistance to OFX and KAN with sensitivities of 67.3% and 76.5%, respectively, and specificities of 100.0%. Therefore, MAS-PCR assays can be used for rapid detection of second-line drug resistance among MDR-TB in China, enabling early administration of appropriate treatment regimens to the affected MDR-TB patients.

Objective: To investigate the drug resistance characteristics of Mycobacterium tuberculosis (MTB) isolates from patients with tuberculosis to 12 antituberculous drugs in China. Methods: All clinical isolates of MTB were isolated from patients with tuberculosis in Shanghai Pulmonary Hospital (SPH) during the period from January 1st to December 31th, 2018. Drug susceptibility testing (DST) was performed in micro plates with 12 antituberculous drugs in accordance with relevant guideline. Demographic information, including sex, age, and treatment history was recorded. Results: A total of 1,950 MTB isolates were included in this retrospective study which were isolated from 1,950 patients from 29 regions in China. One thousand six hundred and forty-four were initial treated and 306 were re-treated in the hospital. Two hundred and eight (10.67%, 208/1,950) cases were diagnosed as multidrug-resistant tuberculosis (MDR-TB), from which 74 (4.50%, 74/1,644) cases were initial treated, and the remaining (43.79%, 134/306) were re-treated cases. Besides, the percentage of extensively drug-resistant tuberculosis (XDR-TB) varied in such 3 different groups: 1.64% (32/1,950) in total cases, 0.30% (5/1,644) in initial treated cases and 8.82% (27/306) in re-treated cases. The total resistance rates were as follows: isoniazid (361, 18.51%), streptomycin (302, 15.49%), rifampin (241, 12.36%), ofloxacin (239, 12.26%), moxifloxacin (232, 11.90%), rifabutin (195, 10.00%), ethambutol (100, 5.13%), cycloserine (55, 2.82%), kanamycin (48, 2.46%), ethionamide (40, 2.05%), amikacin (39, 2.00%), and aminosalicylic acid (21, 1.08%). Rates of resistance to any drug in re-treated cases were significantly higher than in initial treated cases. The drug resistance rates of the 12 drugs were higher in males than in females. Patients older than 60 years had significantly lower percentages of MDR/XDR-TB (7.11 and 0.65%) than in younger age groups. The proportion of re-treated cases in Shanghai (11.38%, 88/773) was lower than that in other regions. Meanwhile, the percentages of MDR/XDR-TB in Shanghai (4.79 and 0.65%) were significantly lower than in other regions. Conclusions: In this study, we found higher proportion of MDR/XDR-TB among re-treated cases than initial treated cases in China and the drug resistance rate of tuberculosis varied with age, sex, and region, indicating that standardized anti-tuberculosis treatment can reduce the incidence of drug-resistant tuberculosis and the recurrence of tuberculosis.

Background: Tuberculosis (TB) and nontuberculous mycobacteriosis are serious threats to health worldwide. A simple non-sequencing method is needed for rapid diagnosis, especially in less experienced hospitals, but there is no specific biomarker commonly used for all mycobacteria. The ku gene of the prokaryotic error-prone non-homologous end joining system (NHEJ) has the potential to be a highly specific detection biomarker for mycobacteria. Methods: A total of 7294 mycobacterial genomes and 14 complete genomes of other families belonging to Corynebacteriales with Mycobacteriaceae were downloaded and analyzed for the existence and variation of the ku gene. Mycobacterium tuberculosis complex (MTBC) and non-tuberculosis mycobacteria (NTM)- specific primers were designed and the actual amplification and identification efficiencies were tested with 150 strains of 40 Mycobacterium species and ten kinds of common respiratory pathogenic bacteria. Results: The ku gene of the NHEJ system was ubiquitous in all genome sequenced Mycobacterium species and absent in other families of Corynebacteriales. On the one hand, as a single gene non-sequencing biomarker, its specific primers could effectively distinguish mycobacteria from other bacteria, MTBC from NTM, which would make the clinical detection of mycobacteria easy and have great clinical practical value. On the other hand, the sequence of ku gene can effectively distinguish NTM to species level with high resolution. Conclusion: The Ku protein existed before the differentiation of Mycobacterium species, which was an important protein involved in maintaining of the genome’s integrity and related to the special growth stage of mycobacteria. It was rare in prokaryotes. These features made it a highly special differential biomarker for Mycobacterium.

Mycobacterium tuberculosis Beijing genotype strains are widespread globally. However, there has been no systematic study on the association between Beijing genotype and the characteristics of drug resistance. In this study, 359 M. tuberculosis isolates from south China were collected and their background information, genotype diversity and drug resistance was investigated. The results revealed that 66.0% of strains (237/359) were categorised as Beijing genotype. There was no statistical difference between Beijing and non-Beijing genotype strains in terms of patient sex, age, place of residence and treatment history. Drug resistance testing showed that 34.8% (125/359) of isolates were resistant to at least one of the seven drugs tested. The proportions of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis were 17.0% and 1.4%, respectively. Previously treated patients presented a significantly higher risk of developing drug resistance than new cases. Although the prevalence of drug resistance was higher in Beijing genotype than in non-Beijing genotype strains, there was no significant difference between these two genotypes in the multivariate analysis. Even in re-treated patients, the association of Beijing genotype with drug resistance was not significant. This study provides an insight into genotype diversity and demonstrates the characteristics of drug resistance in Beijing genotype strains, which will be useful in generating efficient tuberculosis prevention and control strategies in China.

Clofazimine (CFZ) is a promising candidate drug for use in the management of multidrug-resistant tuberculosis (MDR-TB) patients. In this study, the minimum inhibitory concentration (MIC) method and checkerboard method were used to investigate potential synergies between CFZ and moxifloxacin (MOX) or capreomycin (CAP). Thirty Mycobacterium tuberculosis strains were collected, including 13 MDR strains, 2 extensively drug-resistant (XDR) strains, 3 pan-sensitive strains and 12 strains resistant to other drugs. When the minimum fractional inhibitory concentration indexes (FICIs) were calculated, synergy was found in 21 (70.00%) M. tuberculosis strains against the CFZ/CAP combination and 29 (96.67%) against the CFZ/MOX combination. When the maximum FICIs were calculated, 10 of 15 MDR/XDR strains and 2 of 15 other drug-resistant or pan-sensitive strains showed antagonism against the CFZ/CAP combination, whilst 8 of 15 MDR/XDR strains and 1 of 15 other drug-resistant or pan-sensitive strains showed antagonism against the CFZ/MOX combination, respectively. In conclusion, these findings demonstrate that the combination of CFZ and MOX shows better synergism than the combination of CFZ and CAP. The MDR/XDR isolates are more likely to show antagonism than the other drug-resistant or pan-sensitive strains in both the CFZ/MOX and CFZ/CAP combinations. CFZ in combination with MOX may be a promising drug regimen for the treatment of MDR-TB, particularly for susceptible M. tuberculosis infections.

China is one of the 22 countries with high TB burden worldwide, and Sichuan contained the second-largest number of TB cases among all of the Chinese provinces. But the characteristics ofMycobacterium tuberculosiscirculated in Zigong, Sichuan, were still unknown. To investigate the character and drug resistance profile, 265 clinical isolates were cultured from tuberculosis patient’s sputum samples in the year of 2010, of which the genetic profile was determined by using Spoligotyping and MIRU-VNTR typing methods, and the drug sensibility testing to the four first-line and four second-line antituberculosis (anti-TB) drugs was performed by using proportion method on Lowenstein-Jensen (L-J) media. The major Spoligotype was Beijing family (143/265, 53.96%), followed by T (80/265, 30.19%) and H (9/265, 3.40%) genotypes; the total Hunter-Gaston discrimination index (HGDI) of the 24 loci MIRU-VNTR was 0.9995. About 27.17% (72/265) of the isolates were resistant to at least one of the eight tested anti-TB drugs, and for Beijing and non-Beijing family isolates the proportion of drug resistance was 28.47% (41/144) and 25.62% (31/121), respectively. That is, the most prevalent genotype here was Beijing family, and the 24 loci VNTR analysis could supply a high resolution for genotyping, and Beijing and non-Beijing isolates had no difference (p>0.05) for drug resistance.

Objectives. Evaluating the activity of nineteen β-lactams in combination with different β-lactamase inhibitors to determine the most potent combination against Mycobacterium tuberculosis (MTB) in vitro. Methods. Drug activity was examined by drug susceptibility test with 122 clinical isolates from China. Mutations of blaC and drug targets ldtMt1, ldtMt2, dacB2, and crfA were analyzed by nucleotide sequencing. Results. Tebipenem (TBM) in combination with clavulanate (CLA) exhibited the highest anti-TB activity. The MIC of β-lactam antibiotics was reduced most evidently in the presence of CLA, compared to avibactam (AVI) and sulbactam (SUB). Eight polymorphism sites were identified in blaC, which were not associated with β-lactams resistance. Interestingly, one strain carrying G514A mutation in blaC was highly susceptible to β-lactams regardless of the presence of inhibitors. The transpeptidase encoding genes, ldtMt1, ldtMt2, and dacB2, harboured three mutations, two mutations, and one mutation, respectively, but no correlation was found between these mutations and drug resistance. Conclusion. The activity of β-lactams against MTB and different synergetic effect of β-lactamase inhibitors were indicated. TBM/CLA exhibited the most activity and has a great prospect in developing novel anti-TB regimen; however, further clinical research is warranted. Moreover, the resistance to the β-lactam antibiotics might not be conferred by single target mutation in MTB and requires further studies.

The EmbCAB proteins have been considered a target for ethambutol (EMB). Mutations in embCAB are known to confer most EMB resistance. However, the knowledge about the effects of embCAB mutations on the EMB resistance level and about the role of mutation-mutation interactions is limited in China. Here, we sequenced embCAB among 125 Mycobacterium tuberculosis isolates from China and quantified their EMB MICs by testing growth at 10 concentrations. Furthermore, a multivariate regression model was established to assess the effects of both individual mutations and multiple mutations. Our results revealed that in China, 82.6% of EMB-resistant isolates (71/86 isolates) harbored at least one mutation within embCAB . Most of the mutations were located in the embB and embA upstream region. Several individual mutations and multiple mutations within this region contributed to the different levels of EMB resistance. Their effects were statistically significant. Additionally, there was an association between high-level EMB resistance and multiple mutations.

Summary Objectives Mixed infections of Mycobacterium tuberculosis strains have attracted more attention due to their increasing frequencies worldwide, especially in the areas of high tuberculosis (TB) prevalence. In this study, we accessed the rates of mixed infections in a setting with high TB prevalence in Inner Mongolia Autonomous Region of China. Methods A total of 384 M. tuberculosis isolates from the local TB hospital were subjected to mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing method. The single clones of the strains with mixed infections were separated by subculturing them on the Lowenstein–Jensen medium. Results Of these 384 isolates, twelve strains (3.13%) were identified as mixed infections by MIRU-VNTR. Statistical analysis indicated that demographic characteristics and drug susceptibility profiles showed no statistically significant association with the mixed infections. We further subcultured the mixed infection strains and selected 30 clones from the subculture for each mixed infection. Genotyping data revealed that eight (8/12, 66.7%) strains with mixed infections had converted into single infection through subculture. The higher growth rate was associated with the increasing proportion of variant subpopulation through subculture. Conclusions In conclusion, by using the MIRU-VNTR method, we demonstrate that the prevalence of mixed infections in Inner Mongolia is low. Additionally, our findings reveal that the subculture changes the population structures of mixed infections, and the subpopulation with higher growth rate show better fitness, which is associated with high proportion among the population structure after subculture. This study highlights that the use of clinical specimens, rather than subcultured isolates, is preferred to estimate the prevalence of mixed infections in the specific regions.

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon ( embC , embA , and embB ). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB ( embB 306), followed by embB 406, embA (−16), and embB 497. Mutations at embB 306 were associated with EMB resistance. DNA sequencing of embB 306–497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB 306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.

The emergence of extensively drug-resistant tuberculosis (XDR-TB) in China is a great threat to TB control. To determine the molecular characterisation of XDR-TB isolates from China and the correlations between specific drug resistance-associated mutations and different genotype strains, 58 XDR-TB isolates were sequenced in eight drug loci, including katG, inhA, oxyR-ahpC intergenic region, rpoB, eis, rrs, gyrA and gyrB, and were genotyped using spoligotyping and analysis of the noise transfer function region. Compared with the phenotypic data, the sensitivities and specificities for DNA sequencing were 87.9% and 100.0% for isoniazid (INH), 91.4% and 98.3% for rifampicin (RIF), 60.4% and 100.0% for kanamycin (KAN) and 81.0% and 100.0% for ofloxacin (OFX), respectively. A combination of eight drug loci predicted XDR-TB phenotypes with 53.4% sensitivity (31/58 isolates) and 100.0% specificity. The most frequent mutations among these XDR-TB isolates were katG315 and inhA-15 (for INH), 531, 526 and 516 in rpoB (for RIF), rrs1401 and eis-10 (for KAN) and 94, 90 and 91 in gyrA (for OFX). Also, among these XDR-TB isolates, 44 (75.9%) were identified as Beijing genotype strain, of which 31 (70.5%) belonged to the modern Beijing sublineage. inhA-8, rpoB526 and rpoB531 mutations demonstrated significant statistical associations with ancient and modern Beijing family sublineage (P0.05). However, Beijing and non-Beijing genotypes showed no association with specific resistance-conferring mutations. These results will be helpful in designing new molecular biology-based techniques to diagnose XDR-TB in China.

Objectives.We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinicalM. tuberculosisisolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and theVNTR-15Chinalocus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary.

Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 162 clinical M. tuberculosis complex (MTBC) isolates from China, amplified gene encoding Rv3878 and compared the sequences. The results showed that Rv3878, a conserved hypothetical alanine rich protein, is not conserved in M. tuberculosis strains and there are polymorphisms existing in the protein. The large number of amino acid changes in its T cell epitopes may reflect ongoing immune evasion.

Recently, tandem repeat typing has emerged as a rapid and easy method for the molecular epidemiology of the Mycobacterium tuberculosis (M. tuberculosis) complex. In this study, a collection of 19 VNTRs incorporating 15 previously described loci and 4 newly evaluated markers were used to genotype 206 Chinese M. tuberculosis isolates and 9 BCG strains. The discriminatory power was evaluated and compared with that obtained by Spoligotyping. It turned out that 15-locus VNTR could be very useful in M. tuberculosis complex strains genotyping in China. The 4 newly evaluated loci were proved informative and could be useful for future epidemiology studies, especially in Beijing family strains. In addition, a unique pattern of the latter 4 loci were found in Chinese BCG strains.

Pulmonary diseases caused by nontuberculousmycobacteria(NTM) are increasing in incidence and prevalence worldwide. In this study, we identified NTM species of the clinical isolates from 8 provinces in China, in order to preliminarily provide some basic scientific data in the different species and distribution of NTM related to pulmonary disease in China. A total of 523 clinical isolates from patients with tuberculosis (TB) diagnosed clinically from 2005 to 2012 were identified to the species using conventional and molecular methods, including multilocus PCR,rpoBandhsp65PCR-PRA,hsp65,rpoB, and16S-23Sinternal transcribed spacer region sequencing. The isolates were identified into 3 bacterium genera, including NTM,Gordonia bronchialis,andNocardia farcinica, and, for the 488 NTM isolates, 27 species were identified. For all the 27 species of NTM which were found to cause pulmonary infections in humans, the most prevalent species wasM. intracellulare, followed byM. aviumandM. abscessus. And seven other species were for the first time identified in patients with TB in China. NTM species identification is very important for distinguishing between tuberculosis and NTM pulmonary diseases, and the species diversity drives the creation of diverse and integrated identification methods with higher accuracy and efficacy.

Mycobacterium tuberculosis Beijing genotype originated in China and has undergone a dramatic population growth and global spread in the last century. Here, a collection of M. tuberculosis Beijing family isolates from different provinces across all China was genotyped by high-resolution (24-MIRU-VNTR) and low-resolution, high-rank (modern and ancient sublineages) markers. The molecular profiles and global and local phylogenies were compared to the strain phenotype and patient data. The phylogeographic patterns observed in the studied collection demonstrate that large-scale (but not middle/small-scale) distance remains one of the decisive factors of the genetic divergence of M. tuberculosis populations. Analysis of diversity and network topology of the local collections appears to corroborate a recent intriguing hypothesis about Beijing genotype originating in South China. Placing our results within the Eurasian context suggested that important Russian B0/W148 and Asian/Russian A0/94-32 epidemic clones of the Beijing genotype could trace their origins to the northeastern and northwestern regions of China, respectively. The higher clustering of the modern isolates in children and lack of increased MDR rate in any sublineage suggest that not association with drug resistance but other (e.g., speculatively, virulence-related) properties underlie an enhanced dissemination of the evolutionarily recent, modern sublineage of the Beijing genotype in China.

Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family of Mycobacteriaceae and attracts excessive immune responses which cause pathology of the lungs in active tuberculosis. The lack of more sensitive and effective diagnosis reagents advocates a further recognition for the fast diagnostic and immunological measures for tuberculosis. Here, two 12-mer peptides with core sequences of SVSVGMKPSPRP (CS1) and TMGFTAPRFPHY (CS2) were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target. Enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay verified that positive phages exhibited strong binding affinity to mixed tuberculosis-positive serum. BLAST analysis showed that the two sequences may be mimotopes of the Mycobacterium tuberculosis . The diagnostic potential for two synthetic mimotope peptides CS1 and CS2 was evaluated using different panels of serum samples ( n = 181) by ELISA, and the diagnostic parameters were calculated. CS1 and CS2 achieved sensitivity of 89.41% and 85.88%, and specificities were 90.63% and 87.50%, respectively. We hypothesized that the diagnostic based on CS1 and CS2 may become a promising strategy to enhance the detection of Mycobacterium tuberculosis infection due to higher specificity and sensitivity. Therefore, CS1 and CS2 may possess potentials to provide an experimental basis for the diagnosis of tuberculosis.

To treat nontuberculous mycobacteria (NTM) infections more optimally, further research pertaining to mycobacterial susceptibility to antimicrobial agents is required. A total of 82 species of NTM reference strains and 23 species of NTM clinical isolates were included. Minimum inhibitory concentrations (MICs) for 41 drugs were determined using the microdilution method in cation-adjusted Mueller-Hinton broth. The results showed that most of the NTM were susceptible to aminoglycosides, quinolones, three macrolides (clarithromycin, azithromycin and roxithromycin), cefmetazole, linezolid and capreomycin. Rapidly growing mycobacterium strains were additionally susceptible to cefoxitin, clofazimine, rifapentine, doxycycline, minocycline, tigecycline, meropenem and sulfamethoxazole, whereas slowly growing mycobacterium strains were additionally susceptible to rifabutin. This study on the susceptibility of NTM includes the largest sample size of Chinese clinical isolates and reference strains. NTM species-specific drug susceptibility patterns suggested that it is urgent to identify the species of NTM, to normalise the treatment of NTM infectious disease and to clarify the resistance mechanisms of NTM.

Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently.Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs,M. bovisAF2122/97, andM. tuberculosisH37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency.Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development.

Genotyping results and DNA sequencing analysis of 235 Mycobacterium tuberculosis (M. tuberculosis) isolates from China indicated that mutations at codon 995 (Pro CCG to Pro CCA) and 701 (Ile ATT to Thr ACT) in lysX gene (Rv1640c), are specific markers for Beijing and modern Beijing strains, respectively. This observation was also confirmed by 24 genomes of M. tuberculosis strains from other countries. Moreover, a simple and fast multiplex allele-specific PCR (MAS-PCR) method for detecting mutations at codon 995 and 701 in lysX has been established and used to screen 235 DNA samples obtained from M. tuberculosis isolates. In all cases, Beijing and modern Beijing strains were identified correctly.

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

We investigated the spectrum and frequency of mutations in rpsL, rrs, and gidB among 140 multidrug-resistant tuberculosis (MDR-TB) clinical isolates from China. The association between mutations and different genotypes was also analyzed. Our data revealed that 65.7% of MDR-TB were resistant to streptomycin (STR), and 90.2% of STR-resistant isolates were Beijing strains. STR resistance was correlated with Beijing family (P=0.00). Compared with phenotypic data, detection of mutations for the combination of these 3 genes exhibited 94.6% sensitivity, 91.7% specificity, and 93.6% accuracy. The most common mutations in STR-resistant isolates were rpsL128, 262, and rrs514, of which rpsL128 showed association with Beijing lineage (P=0.00). A combination of these 3 mutations can serve as the reliable predictors for STR resistance, showing the sensitivity, specificity, and accuracy of 85.9%, 97.9%, and 90.0%, respectively. Furthermore, gidBA276C, not A615G, was Beijing lineage specific. These findings are useful to develop rapid molecular diagnostic methods for STR resistance in China.

Summary Objective To evaluate the performance of three interferon-gamma release assay (IGRA) kits in detecting Mycobacterium tuberculosis infection in China. Methods A multicenter clinical trial was used to compare the effectiveness and application of the three kits. A total of 1026 participants were enrolled at three hospitals, including 597 tuberculosis (TB) patients diagnosed clinically (517 patients with pulmonary TB and 80 patients with extrapulmonary TB) and 429 negative controls (244 patients with pulmonary disease but not TB, or with non-tuberculosis mycobacterial lung diseases, and 185 healthy people). Detection performance indicators including sensitivity, specificity, and the Youden index (YI) were used to evaluate performance. Results Through bacterial culture evaluation, 224 of the 517 pulmonary TB patients were positive and all 429 negative controls were negative. When the gold standard bacterial methods were used, the sensitivity, specificity, and YI were 89.7% (201/224), 91.1% (391/429), and 0.81 for T-SPOT.TB, 86.2% (193/224), 87.2% (374/429), and 0.73 for QB-SPOT, and 83.9% (188/224), 88.6% (380/429), and 0.73 for TB-IGRA, respectively. There were no significant differences in the sensitivity and specificity of the three kits. Conclusions The results showed that the three kits had very high sensitivity and specificity and exhibited a good performance for the detection of M. tuberculosis infection.

There is an urgent need for new immunodominant antigens to improve the diagnosis of tuberculosis (TB) and the efficacy of the TB vaccine to control the disease worldwide. In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (MTB)-specific antigen, Rv2351c, from region of difference (RD) 7 of the MTB genome, and investigated the potency of the vaccine by identifying its immunological function in human and animal immunological experiments. Twenty T-cell epitopes were identified using TEpredict and prediction tools from the Immune Epitope Database and Analysis Resource. A total of 159 subjects, including 61 patients with pulmonary TB, 38 patients with no TB and 55 healthy donors, were recruited and analyzed with an enzyme-linked immunospot (ELISpot) assay. The ELISpot assay using Rv2351c to detect TB infection, as compared with bacteriological tests as the gold standard, had a sensitivity and specificity of 61.4% (35/57) and 91.4% (85/93), respectively. The ELISpot assay using Rv2351c had a good conformance (κ=0.554) as compared with the bacteriological test. Rv2351c also elicited a potent cellular immune response with a high expression of cytokines (IFN-γ (4978±596.7 μg/mL) and IL-4 (68.3±15.5 μg/mL)) and a potent humoral immune response with a high concentration of IgG (1:2.2 × 106), IgG1 (1:4.5 × 105) and IgG2a (1:1.6 × 106) in immunized BALB/c mice. In addition, the ratio of IgG2a/IgG1 indicated that Rv2351c induced cellular immunity in the mice. The results of this study indicated that Rv2351c is an antigen with good immunogenicity that may potentially be used to develop diagnostic techniques and new TB vaccines.

To identify the mutations in multi- and extensively drug-resistant tuberculosis isolates and to evaluate the use of molecular markers of resistance, we analyzed 257 multi- and extensively drug-resistant isolates and 64 pan-sensitive isolates from 23 provinces in China. Seven loci associated with drug resistance, including rpoB for rifampin (RIF), katG, inhA and oxyR-ahpC for isoniazid (INH), gyrA and gyrB for ofloxacin (OFX), and rrs for kanmycin (KAN), were examined by DNA sequencing. Compared with the phenotypic data, the sensitivity and specificity for DNA sequencing were 91.1% and 98.4% for RIF, 80.2% and 98.4% for INH, 72.2% and 98.3% for OFX and 40% and 98.2% for KAN, respectively. The most common mutations found in RIF, INH, OFX and KAN resistance were Ser531Leu (48.2%) in rpoB, Ser315Thr (49.8%) in katG, C(-15)T (10.5%) in inhA, Asp94Gly (20.3%), Asp94Ala (12.7%) and Ala90Val (21.5%) in gyrA, and A1401G (40%) in rrs. This molecular information will be helpful to establish new molecular biology-based methods for diagnosing multi- and extensively drug-resistant tuberculosis in China.

In our previous study, we found that polymorphisms of the mpt64 gene in the Mycobacterium tuberculosis complex (MTBC) may be the reason for changes in the antigen produced, which may in turn cause alterations of related functions. Based on the data, we further analyzed T/B cell epitope regions and non-epitope regions and compared amino acid changes between them. It turned out the polymorphisms of MPT64 affected both T cell epitopes and B cell epitopes, which thus influenced cell-mediated immunity and humoral immunity at the same time. Both T cell epitope regions and B cell epitope regions had higher dN/dS values than corresponding non-epitope regions, which means the formers have accumulated significantly more amino acid changes than the latters.