Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Impact of MSMEG5257 Deletion on Mycolicibacterium smegmatis Growth.

Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. Mycolicibacterium smegmatis (Ms) msmeg5257 is a hemolysin III family protein that is homologous to Mycobacterium tuberculosis (Mtb) Rv1085c, but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δmsmeg5257 strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of msmeg5257 in Ms growth. The correlative analysis showed that the deletion of msmeg5257 inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that msmeg5257 plays a role in maintaining Ms metabolic homeostasis, and the deletion of msmeg5257 significantly impacts the growth rate of Ms. Furthermore, msmeg5257, a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.

Proficiency testing for drug susceptibility testing of Mycobacterium tuberculosis complex using commercial broth microdilution plate in China in 2021.

OBJECTIVES: The characteristic and performance of Broth microdilution (BMD) plates for drug susceptibility of Mycobacterium tuberculosis have not been systematically evaluated in China. This study was designed to review the key information and assess the performance of BMD plates by analysis of proficiency testing results. METHODS: We retrospectively analysed the proficiency testing results of phenotypic drug susceptibility testing (PT-DST) of 45 laboratories using BMD plates in China in 2021. Critical information, such as drug layout, concentration range of each drug, plate storage conditions and duration, operating procedures, and interpretation criteria for binary results were compared. The performance was also analysed. RESULTS: Eight types of BMD plates produced by four manufactures were reported. The drug layout, number of drugs on plates, and concentration range varied a lot between different plates. The total sensitivity and specificity of BMD plates for drug susceptibility of Mycobacterium tuberculosis to ten drugs (isoniazid (INH), rifampin (RIF), kanamycin (KAM), amikacin (AM), levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ), and delamanid (DLM)) were 93.9% (95% CI 92.-94.9) and 99.1% (95% CI 98.8-99.3), respectively. The lowest sensitivity was 84.8% (95% CI 80.3-88.4) for LFX and 86.4% (95% CI 82.5-89.6) for MFX, or 87.5% (95% CI 84.2-90.2) for Y1 plate and 87.9% (95% CI 83.5-91.1) for T plate. The lowest specificity was 94.4% (95% CI 91.4-96.4) for DLM, or 97.9% (95% CI 96.8-98.7) for B3 plate. CONCLUSION: Commercial BMD plates in China showed varied drug layouts and operational procedures, indicating the urgency of standardization. The lower performance for some drugs showed the low quality of the plates utilized or lack of proficiency of lab staffs in operating and interpreting results.

Diagnostic efficacy of an optimized nucleotide MALDI-TOF-MS assay for anti-tuberculosis drug resistance detection.

PURPOSE: We aimed at evaluating the diagnostic efficacy of a nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay to detect drug resistance of Mycobacterium tuberculosis. METHODS: Overall, 263 M. tuberculosis clinical isolates were selected to evaluate the performance of nucleic MALDI-TOF-MS for rifampin (RIF), isoniazid (INH), ethambutol (EMB), moxifloxacin (MXF), streptomycin (SM), and pyrazinamide (PZA) resistance detection. The results for RIF, INH, EMB, and MXF were compared with phenotypic microbroth dilution drug susceptibility testing (DST) and whole-genome sequencing (WGS), and the results for SM and PZA were compared with those obtained by WGS. RESULTS: Using DST as the gold standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 98.2%, 98.7%, and 0.97 for RIF; 92.8%, 99%, and 0.90 for INH; 82.4%, 98.0%, and 0.82 for EMB; and 92.6%, 99.5%, and 0.94 for MXF, respectively. Compared with WGS as the reference standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 97.4%, 100.0%, and 0.98 for RIF; 98.7%, 92.9%, and 0.92 for INH; 96.3%, 100.0%, and 0.98 for EMB; 98.1%, 100.0%, and 0.99 for MXF; 98.0%, 100.0%, and 0.98 for SM; and 50.0%, 100.0%, and 0.65 for PZA. CONCLUSION: The nucleotide MALDI-TOF-MS assay yielded highly consistent results compared to DST and WGS, suggesting that it is a promising tool for the rapid detection of sensitivity to RIF, INH, EMB, and MXF.

Association of lineage 4.2.2 of Mycobacterium tuberculosis with the 63-bp deletion variant of the mpt64 gene.

IMPORTANCE: To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.

Insight into the drug-resistant characteristics and genetic diversity of multidrug-resistant Mycobacterium tuberculosis in China.

Multidrug-resistant tuberculosis (MDR-TB) has a severe impact on public health. To investigate the drug-resistant profile, compensatory mutations and genetic variations among MDR-TB isolates, a total of 546 MDR-TB isolates from China underwent drug-susceptibility testing and whole genome sequencing for further analysis. The results showed that our isolates have a high rate of fluoroquinolone resistance (45.60%, 249/546) and a low proportion of conferring resistance to bedaquiline, clofazimine, linezolid, and delamanid. The majority of MDR-TB isolates (77.66%, 424/546) belong to Lineage 2.2.1, followed by Lineage 4.5 (6.41%, 35/546), and the Lineage 2 isolates have a strong association with pre-XDR/XDR-TB (P < 0.05) in our study. Epidemic success analysis using time-scaled haplotypic density (THD) showed that clustered isolates outperformed non-clustered isolates. Compensatory mutations happened in rpoA, rpoC, and non-RRDR of rpoB genes, which were found more frequently in clusters and were associated with the increase of THD index, suggesting that increased bacterial fitness was associated with MDR-TB transmission. In addition, the variants in resistance associated genes in MDR isolates are mainly focused on single nucleotide polymorphism mutations, and only a few genes have indel variants, such as katG, ethA. We also found some genes underwent indel variation correlated with the lineage and sub-lineage of isolates, suggesting the selective evolution of different lineage isolates. Thus, this analysis of the characterization and genetic diversity of MDR isolates would be helpful in developing effective strategies for treatment regimens and tailoring public interventions. IMPORTANCE Multidrug-resistant tuberculosis (MDR-TB) is a serious obstacle to tuberculosis prevention and control in China. This study provides insight into the drug-resistant characteristics of MDR combined with phenotypic drug-susceptibility testing and whole genome sequencing. The compensatory mutations and epidemic success analysis were analyzed by time-scaled haplotypic density (THD) method, suggesting clustered isolates and compensatory mutations are associated with MDR-TB transmission. In addition, the insertion and deletion variants happened in some genes, which are associated with the lineage and sub-lineage of isolates, such as the mpt64 gene. This study offered a valuable reference and increased understanding of MDR-TB in China, which could be crucial for achieving the objective of precision medicine in the prevention and treatment of MDR-TB.

Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis.

Objectives: The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Methods: Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. Results: The sensitivity of the MLP-RAP assay could reach 5 copies/µl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/µl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. Conclusion: MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.

Endogenous relapse and exogenous reinfection in recurrent pulmonary tuberculosis: A retrospective study revealed by whole genome sequencing.

Background: Tuberculosis may reoccur due to reinfection or relapse after initially successful treatment. Distinguishing the cause of TB recurrence is crucial to guide TB control and treatment. This study aimed to investigate the source of TB recurrence and risk factors related to relapse in Hunan province, a high TB burden region in southern China. Methods: A population-based retrospective study was conducted on all culture-positive TB cases in Hunan province, China from 2013 to 2020. Phenotypic drug susceptibility testing and whole-genome sequencing were used to detect drug resistance and distinguish between relapse and reinfection. Pearson chi-square test and Fisher exact test were applied to compare differences in categorical variables between relapse and reinfection. The Kaplan-Meier curve was generated in R studio (4.0.4) to describe and compare the time to recurrence between different groups. p < 0.05 was considered statistically significant. Results: Of 36 recurrent events, 27 (75.0%, 27/36) paired isolates were caused by relapse, and reinfection accounted for 25.0% (9/36) of recurrent cases. No significant difference in characteristics was observed between relapse and reinfection (all p > 0.05). In addition, TB relapse occurs earlier in patients of Tu ethnicity compared to patients of Han ethnicity (p < 0.0001), whereas no significant differences in the time interval to relapse were noted in other groups. Moreover, 83.3% (30/36) of TB recurrence occurred within 3 years. Overall, these recurrent TB isolates were predominantly pan-susceptible strains (71.0%, 49/69), followed by DR-TB (17.4%, 12/69) and MDR-TB (11.6%, 8/69), with mutations mainly in codon 450 of the rpoB gene and codon 315 of the katG gene. 11.1% (3/27) of relapse cases had acquired new resistance during treatment, with fluoroquinolone resistance occurring most frequently (7.4%, 2/27), both with mutations in codon 94 of gyrA. Conclusion: Endogenous relapse is the main mechanism leading to TB recurrences in Hunan province. Given that TB recurrences can occur more than 4 years after treatment completion, it is necessary to extend the post-treatment follow-up period to achieve better management of TB patients. Moreover, the relatively high frequency of fluoroquinolone resistance in the second episode of relapse suggests that fluoroquinolones should be used with caution when treating TB cases with relapse, preferably guided by DST results.

Evaluation Study of xMAP TIER Assay on a Microsphere-Based Platform for Detecting First-Line Anti-Tuberculosis Drug Resistance.

Early diagnosis of drug susceptibility for tuberculosis (TB) patients could guide the timely initiation of effective treatment. We evaluated a novel multiplex xMAP TIER (Tuberculosis-Isoniazid-Ethambutol-Rifampicin) assay based on the Luminex xMAP system to detect first-line anti-tuberculous drug resistance. Deoxyribonucleic acid samples from 353 Mycobacterium tuberculosis clinical isolates were amplified by multiplex polymerase chain reaction, followed by hybridization and analysis through the xMAP system. Compared with the broth microdilution method, the sensitivity and specificity of the xMAP TIER assay for detecting resistance was 94.9% (95%CI, 90.0-99.8%) and 98.9% (95%CI, 97.7-100.0%) for rifampicin; 89.1% (95%CI, 83.9-94.3%) and 100.0% (95%CI, 100.0-100.0%) for isoniazid; 82.1% (95% CI, 68.0-96.3%) and 99.7% (95% CI, 99.0-100.0%) for ethambutol. With DNA sequencing as the reference standard, the sensitivity and specificity of xMAP TIER for detecting resistance were 95.0% (95% CI, 90.2-99.8%) and 99.6% (95% CI, 98.9-100.0%) for rifampicin; 96.9% (95% CI, 93.8-99.9%) and 100.0% (95% CI, 100.0-100.0%) for isoniazid; 86.1% (95% CI, 74.8-97.4%) and 100.0% (95% CI, 100.0-100.0%) for ethambutol. The results achieved showed that the xMAP TIER assay had good performance for detecting first-line anti-tuberculosis drug resistance, and it has the potential to diagnose drug-resistant tuberculosis more accurately due to the addition of more optimal design primers and probes on open architecture xMAP system.

Genomic analysis of diversity, biogeography, and drug resistance in Mycobacterium bovis.

Mycobacterium bovis is the cause of bovine tuberculosis, and it can also cause disease in humans, with symptoms similar to those caused by M. tuberculosis. However, our understanding of its genomic diversity, biogeography, and drug resistance remains incomplete. We performed a comparative and phylogenetic analysis of 3228 M. bovis genomes from 24 countries. Following drug susceptibility testing, we applied a bacterial genome-wide association study to capture associations between genomic variation and drug resistance in 74 newly isolated strains from China. The data show that the cattle-adapted M. bovis were divided into six lineages with a strong phylogeographical population structure. Lineages 1 and 6 are the most widespread globally, while others show a strong geographical restriction. Note that 17.39% of M. bovis isolates were resistant to at least one drug in China. Furthermore, we identify genomic variations associated with an increased risk of resistance acquisition. This study furthers our knowledge of M. bovis diversity, biogeography, and drug resistance and will facilitate more deeply informed genomic tracking and surveillance to minimize its threat to human health, as a cause of zoonotic tuberculosis.

Detection of Mycobacterium tuberculosis Rifampicin Resistance Conferred by Borderline rpoB Mutations: Xpert MTB/RIF is Superior to Phenotypic Drug Susceptibility Testing.

Objective: To compare the ability of detection of borderline rifampicin resistance in Mycobacterium tuberculosis between molecular assay and phenotypic drug susceptibility tests. Methods: Fifty-seven isolates with His445Leu, Asp435Tyr, Leu452Pro, Leu430Pro, His445Asn, Ile491Phe, and His445Ser mutations in rpoB gene identified by whole-genome sequencing conferring borderline rifampicin resistance were included. Molecular-based Xpert MTB/RIF, phenotypic Löwenstein-Jensen (L-J) medium-based drug susceptibility test (DST) with a critical concentration of 40.0µg/mL and minimal inhibitory concentration (MIC) assay were performed to detect borderline rifampicin resistance. Results: When using Xpert MTB/RIF, 48/57 (84.2%) isolates exhibited resistance to rifampicin. 25/57 (43.9%) and 33/57 (57.9%) isolates showed rifampicin resistance by L-J medium-based DST with 4 and 6 weeks of incubation, respectively. 30/57 (52.6%) and 40/57 (70.2%) strains were resistant to rifampicin by MIC method at cutoff values of 1.0 and 0.5µg/mL, respectively. The detection rate of rifampicin resistance of Xpert MTB/RIF was significantly higher than that of phenotypic methods (p < 0.001). Of the 57 isolates with borderline rpoB mutations, 5 (8.8%) had MICs of 0.25 or 0.12µg/mL, 22 (38.6%) had MICs of 0.5µg/mL or 1.0µg/mL, and 30 (52.6%) other isolates showed MICs ≥2.0µg/mL. Conclusion: Molecular-based Xpert MTB/RIF showed superior ability to detect borderline rifampicin resistance over phenotypic DST methods. Extending the incubation time of L-J DST or lowering the cutoff value of the MIC method can improve borderline rifampicin resistance detection.

Diversity of glpK Gene and Its Effect on Drug Sensitivity in Mycobacterium bovis.

Background: Glycerol kinase (glpK) is essential for the first step of glycerol catabolism in Mycobacterium tuberculosis. However, Mycobacterium bovis has been known to grow poorly in glycerol media because of a base insertion in the glpK gene. Methods: We analyzed the glpK gene sequences of 60 clinical M. bovis isolates, and determined the minimum inhibitory concentration of 14 drugs by microdilution method to evaluate the effect of frameshift mutations on drug sensitivity. The effect of M. bovis growth rate on its drug sensitivity was investigated using bacteria grown on glycerol or pyruvate. Results: A total of 44 (73.33%) clinical M. bovis isolates have frameshift mutations in a homopolymeric tract of 7 cytosines in the glpK gene. 15.00% M. bovis isolates showed phenotypic drug resistance. Glycerol metabolism-deficient M. bovis showed reduced susceptibility to 9 out of 14 tested drugs. Mutations in the glpK gene can lead to impaired growth in glycerol-based media, while the minimal inhibitory concentration values of slow-growing M. bovis were higher. Conclusion: Mutations in the glpK gene can lead to slowed growth and reduced susceptibility to drugs in M. bovis, which may contribute to the emergence of drug-resistant M. bovis and pose a threat to human health owing to the zoonotic capacity of M. bovis.

Drug-Resistant Characteristics, Genetic Diversity, and Transmission Dynamics of Rifampicin-Resistant Mycobacterium tuberculosis in Hunan, China, Revealed by Whole-Genome Sequencing.

To gain a deep insight into the additional drug-resistant profiles, genetic diversity, and transmission dynamics of rifampicin-resistant tuberculosis (RR-TB) circulating in Hunan province, drug susceptibility testing and whole-genome-sequencing were performed among RR-TB strains collected from Jan. 2013 to Jun. 2018 in Hunan province. A total of 124 RR-TB strains were recovered successfully and included into the final analysis. Lineage 2.2.1 was the dominant sublineage, accounting for 72.6% (90/124), followed by lineage 4.5 (11.3%, 14/124), lineage 4.4 (8.1%, 10/124), lineage 4.2 (6.5%, 8/124) and lineage 2.2.2 (1.6%, 2/124). Overall, 83.1% (103/124) and 3.2% (4/124) of RR-TB were MDR-TB and XDR-TB, respectively. Nearly 30% of RR-TB isolates were resistant to fluoroquinolones, and 26.6% (33/124) were pre-XDR-TB. Moreover, 30.6% (38/124) of RR-TB strains were identified as phenotypically resistance to pyrazinamide. Totally, 17 clusters containing 48 (38.7%, 48/124) RR-TB strains were identified, ranging in size from 2 to 10 isolates. No significant difference was detected in clustering rate between lineage 2 and lineage 4 (χ2 = 0.027, P = 0.870). Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions. IMPORTANCE Comprehensive information such as genetic background and drug-resistant profile of MTB strains could help to tailor public interventions. However, these data are limited in Hunan province, one of the provinces with high-TB burden in China. So, this study aimed to provide us with deep insight into the molecular epidemiology of RR-TB isolates circulating in Hunan province by combining phenotypic drug susceptibility testing and whole-genome sequencing. To our knowledge, this is the first study to use whole-genome sequencing data of RR-TB strains spanning more than 5 years for molecular epidemiology analysis in Hunan province, which allows us to identify genetic background information and clustered strains more accurately. Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions.

Whole-genome sequencing for surveillance of tuberculosis drug resistance and determination of resistance level in China.

OBJECTIVES: Phenotypic drug susceptibility testing for prediction of tuberculosis (TB) drug resistance is slow and unreliable, limiting individualized therapy and monitoring of national TB data. Our study evaluated whole-genome sequencing (WGS) for its predictive accuracy, use in TB drug-resistance surveillance and ability to quantify the effects of resistance-associated mutations on MICs of anti-TB drugs. METHODS: We used WGS to measure the susceptibility of 4880 isolates to ten anti-TB drugs; for pyrazinamide, we used BACTEC MGIT 960. We determined the accuracy of WGS by comparing the prevalence of drug resistance, measured by WGS, with the true prevalence, determined by phenotypic susceptibility testing. We used the Student-Newman-Keuls test to confirm MIC differences of mutations. RESULTS: Resistance to isoniazid, rifampin and ethambutol was highly accurately predicted with at least 92.92% (95% confidence interval [CI], 88.19-97.65) sensitivity, resistance to pyrazinamide with 50.52% (95% CI, 40.57-60.47) sensitivity, and resistance to six second-line drugs with 85.05% (95% CI, 80.27-89.83) to 96.01% (95% CI, 93.89-98.13) sensitivity. The rpoB S450L, katG S315T and gyrA D94G mutations always confer high-level resistance, while rpoB L430P, rpoB L452P, fabG1 C-15T and embB G406S often confer low-level resistance or sub-epidemiological cutoff (ECOFF) MIC elevation. CONCLUSION: WGS can predict phenotypic susceptibility with high accuracy and could be a valuable tool for drug-resistance surveillance and allow the detection of drug-resistance level; It can be an important approach in TB drug-resistance surveillance and for determining therapeutic schemes.

Strong Increase in Moxifloxacin Resistance Rate among Multidrug-Resistant Mycobacterium tuberculosis Isolates in China, 2007 to 2013.

We designed this study to determine the trend of moxifloxacin resistance among multidrug-resistant tuberculosis (MDR-TB) patients from 2007 to 2013 in China to inform the composition of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB) treatment regimens. We assessed moxifloxacin resistance among MDR-TB isolates collected in national drug resistance surveys in 2007 and 2013 that included 3,634 smear-positive and 7,206 culture-positive pulmonary tuberculosis patients, respectively. Moxifloxacin susceptibility was examined by a Mycobacterium growth indicator tube (MGIT) 960 for the 2007 isolates, and by the minimum inhibitory concentration (MIC) method for the 2013 isolates, at both breakpoints 0.5 and 2.0 µg/mL. Risk factors were explored through multivariable log-binominal regression analysis. Mutations in gyrA and gyrB for part of the isolates were also studied through sequencing. Of 401 MDR strains isolated in 2007, moxifiloxacin resistance could be determined for 319 (79.6%): 41 (12.9%) and 10 (3.1%) were resistant at 0.5 and 2.0 µg/mL, respectively. Of 365 MDR strains isolated in 2013, 338 (92.6%) could be analyzed: 140 (41.4%) and 79 (23.4%) were resistant at 0.5 and 2.0 µg/mL. For patients in 2007, no characteristics were significantly associated with moxifloxacin resistance. For patients in 2013, patients aged ≥60 years (adjusted prevalence ratio [aPR], 1.46; 95% confidence interval [CI], 1.10 to 1.93) were more likely to have resistance at 0.5 µg/mL, whereas those residing in eastern China compared to those in central China had an increased risk of resistance at both 0.5 (aPR, 1.85; 95% CI, 1.38 to 2.48) and 2.0 µg/mL (aPR, 2.14; 95% CI, 1.35 to 3.40). Sequencing results were obtained for 245 and 266 MDR-TB isolates in 2007 and 2013, respectively. In total, 34 of 38 (89.5%) and 89 of 104 (85.6%) of 2007 and 2013 moxifloxacin-resistant (0.5 µg/mL) MDR-TB strains had mutations in the gyrA and gyrB gene, respectively. Asp94Gly was the most common mutation among 2007 (11 of 38, 28.9%) and 2013 isolates (24 of 104, 23.1%) and conferred high-level moxifloxacin resistance. Moxifloxacin resistance among MDR-TB patients in China increased from modest to high from 2007 to 2013. Moxifloxacin should be used carefully as a potentially effective drug for composing MDR/RR-TB regimens especially for elderly patients in China. Individual susceptibility testing especially rapid molecular-based assays should be conducted to confirm the susceptibility to moxifloxacin. IMPORTANCE China is one of the high-burden countries for multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). Moxifloxacin is one of the critical antituberculosis drugs for MDR/RR-TB treatment. Susceptibility to moxifloxacin is therefore very important to compose effective regimens and to provide protection against development of resistance of companion drugs such as bedaquiline and linezolid. There are, however, no nationally representative data on moxifloxacin resistance among MDR/RR-TB cases in China. Therefore, we assessed the resistance prevalence for moxifloxacin among MDR-TB strains isolated in national drug resistance surveys in 2007 and 2013 that covered 72 sites around the country. We demonstrate that the prevalence of moxifloxacin resistance in MDR-TB isolates increased from modest to high, which should prompt the national tuberculosis program to use moxifloxacin cautiously in second-line regimens to treat MDR/RR-TB unless susceptibility can be laboratory-confirmed.

National Proficiency Testing of Molecular Diagnostics for Tuberculosis and Drug Resistance Detection - China, 2014-2019.

What is already known about this topic? Proficiency testing (PT) is a key component of quality assurance and is essential in ensuring accurate laboratory diagnosis of tuberculosis (TB) and drug-resistant TB. The National Tuberculosis Reference Laboratory (NTRL) developed a novel PT panel to test laboratories' proficiency for molecular TB diagnostic assays throughout the TB laboratory network. A total of 6 PT rounds for molecular diagnostics were conducted by NTRL from 2014 to 2019. What is added by this report? PT conducted using artificial sputum specimens increased from 120 in the first round to 1,835 in the sixth round. Overall, laboratories demonstrated good proficiency for MTB and drug-resistance detection by molecular diagnostics, which is evident from the qualification rates over the six rounds: 95%, 97%, 96%, 93%, 93%, and 97%, respectively. What are the implications for public health practice? The use of artificial sputum specimens for PT panel production to test TB molecular diagnostics in China is feasible. Most of the participating laboratories provided reliable molecular diagnostic results for MTB and drug-resistance detection. The TB laboratory network can be instrumental in implementing PT expansion and improving the quality of TB molecular diagnosis in China.

The Epidemiological Characteristics of Pulmonary Tuberculosis - Kashgar Prefecture, Xinjiang Uygur Autonomous Region, China, 2011-2020.

Introduction: National Notifiable Disease Reporting System (NNDRS) plays an important role in the early detection and control of tuberculosis (TB) in China. This study analyzed the epidemiological characteristics of pulmonary tuberculosis (PTB) in Kashgar Prefecture, Xinjiang Uygur Autnomous Region, China from 2011 to 2020 to provide a scientific basis for developing TB control strategies and measures in Kashgar.Methods:The data were collected from the NNDRS, which included the geographical distribution, age, sex, occupation, and pathogenic classification of reported PTB cases in 12 counties/cities of Kashgar Prefecture from 2011 to 2020. Descriptive statistics were used to describe the characteristic of PTB epidemic in Kashgar.Results: There were 189,416 PTB cases reported during 2011-2020, with a mean annual PTB case notification rate (CNR) of 451.29/100,000. A rising trend in the rate of reported PTB between 2011 and 2017 (χ 2 trend=26.09, P<0.01) and a declining trend between 2018 and 2020 (χ 2 trend=314.44, P<0.01) were observed. The months with the highest reported number of PTB cases were March to May and November to December. The mean annual rate of reported PTB was 451.88/100,000 for males and 450.67/100,000 for females. In addition, 19.76% of patients were bacteriologically-confirmed (Bac+) cases (37,425/189,416), and the mean annual Bac+ CNR was 89.17/100,000, rising from 64.76/100,000 in 2011 to 139.12/100,000 in 2020 (χ 2 trend=74.44, P<0.01).Conclusions: The CNR of reported PTB in Kashgar showed a significant declining trend in the past three years. Males, elderly population, winter and spring, and farmers as an occupation were the main factors associated with high incidence of tuberculosis in Kashgar. Targeted prevention and treatment of TB should be strengthened in key groups in this region.