OBJECTIVES:Whole-genome sequencing (WGS) enables comprehensive characterization of Mycobacterium tuberculosis (MTB), but its routine clinical use is limited by the lack of standardized and multicenter-validated analytical workflows. METHODS:We developed TB-Genaly, a user-friendly and secure bioinformatics pipeline for MTB WGS analysis. Performance of drug-resistance prediction was evaluated using a multicenter, population-based cohort comprising 461 clinical MTB isolates from four centers, each with paired phenotypic drug susceptibility testing (pDST) results. Sensitivity and specificity were calculated to assess concordance between genotypic predictions and pDST. RESULTS:TB-Genaly demonstrated high predictive performance for anti-tuberculosis drug resistance. For first-line drugs, it achieved sensitivity and specificity of 95.8% and 92.6% for rifampicin, 95.5% and 92.4% for isoniazid, 91.8% and 99.4% for pyrazinamide, and 87.3% and 92.7% for ethambutol. For second-line drugs, sensitivity and specificity ranged from 87.8% to 94.7% and 97.5% to 98.0%. The mean values of sensitivity and specificity for predicting MDR-TB were 95.4% (95%CI 92.3%-97.7%) and 93.5% (95%CI 91.1-95.5%), respectively. Receiver operating characteristic (ROC) curve analysis further confirmed excellent discriminatory power, with area under the curve (AUC) values of 0.999, 0.997, 0.995 and 0.969 for the four agents. Additionally, TB-Genaly supports automated generation of standardized clinical reports and transmission network visualizations. CONCLUSION:TB-Genaly provides accurate, standardized WGS-based drug-resistance prediction across multiple centers. Its usability and comprehensive reporting facilitate integration into clinical diagnostics and public health surveillance, highlighting its potential to support clinical decision-making and tuberculosis control efforts.

Background:Drug-resistant tuberculosis (DR-TB) remains a major public health challenge in China, yet long-term epidemiological data from key regions such as Hunan Province in South-Central China are still limited. Objective:This study aimed to characterize the epidemiological trends, spatial distribution, and risk factors of Single drug-resistant tuberculosis (SDR-TB), poly-drug-resistant tuberculosis (PDR-TB), multidrug-resistant tuberculosis (MDR-TB), rifampicin-resistant tuberculosis (RR-TB), and isoniazid-resistant tuberculosis (INH-R TB) in Hunan Province between 2014 and 2023, to inform region-specific control strategies. Methods:It was a retrospective analysis that was conducted on 6,597 laboratory-confirmed DR-TB cases. Data were obtained from the Provincial Tuberculosis Control Institute. All patients underwent phenotypic drug susceptibility testing. Independent risk factors for DR-TB subtypes were identified through multivariable logistic regression, with adjusted odds ratios (ORs) and corresponding 95% confidence intervals (CIs) calculated. Results:A total of 6,597 patients with DR-TB were included in this 10-year analysis. Among them, 74.97% were male and 64.44% were farmers. The highest case burden was observed in the 50-59 age group (24.78%). Spatially, cases clustered mainly in the Changsha (16.39%), Shaoyang (13.78%), and Loudi (9.05%). The most common resistance subtypes were INH-R TB (56.27%) and MDR-TB (52.37%). The distribution of all DR-TB subtypes varied significantly across age groups (p < 0.05), with peaks in middle-aged and older adults. Over time, the detection rate of MDR-TB was highest in 2018, while RR-TB remained the most frequently detected resistance type. Multivariable analysis identified significant regional and demographic disparities. The eastern region of Hunan was associated with an increased risk of SDR-TB (OR = 1.334) and PDR-TB (OR = 1.208), whereas the western region carried the highest risk for MDR-TB (OR = 1.734). Female patients consistently showed lower risks of MDR-TB (OR = 0.819) and RR-TB (OR = 0.784) compared with males. Conclusion:This study presents a 10-year epidemiological assessment of DR-TB in Hunan Province, China, covering 2014-2023. A disproportionately high burden was observed among middle-aged and older male farmers. The predominance of INH-R TB and MDR-TB, together with distinct regional and demographic risk profiles, underscores an urgent need to strengthen TB control measures. These results support the implementation of targeted interventions, including intensified screening in high-risk populations and in high-incidence areas, along with optimized treatment regimens, to curb the ongoing DR-TB epidemic in South-Central China.

Background There is widespread use of Xpert MTB/RIF for tuberculosis (TB) diagnosis and rifampin resistance (RIF-R) detection. Despite this, concerns have been raised about false-positive results, which are more prevalent in patients with very low bacterial loads. Methods We performed a retrospective analysis of 717 patients at Hunan Chest Hospital from January 2022 to January 2023, all of whom had Xpert MTB/RIF results indicating very low bacterial loads. The accuracy of Xpert in diagnosing TB was evaluated against a composite reference standard (CRS), which classified cases via symptom, liquid culture, imaging assessments, immunological tests, and therapeutic response. In addition, rifampicin resistance was adjudicated using a composite reference standard of pDST and rpoB sequencing, with pDST as the primary reference for Xpert MTB/RIF results and sequencing used to resolve discrepancies. Results Of 717 patients with Xpert detected very low bacterial loads, 175 (24.4%) yielded positive cultures, while 16 cases identified as non-tuberculous mycobacteria (NTM). The positive predictive value (PPV) of Xpert for bacteriologically confirmed TB was 90.8% (159/175). For 542 culture-negative patients, Xpert's PPV was 81.7% (443/542) in clinically diagnosed TB (80.4% pulmonary TB, 88.9% extrapulmonary TB). In 121 patients with pDST, Xpert for RIF-R had 68.4% sensitivity (13/19), 88.2% specificity (90/102), respectively. Among the 6 patients who were RIF-R by pDST but rifampin susceptible by Xpert, four cases harbored mutations inside the 81-bp Rifampicin Resistance-Determining Region (RRDR) region of rpoB. Conclusion Xpert demonstrates good performance in diagnosing TB among the patients with results showing very low bacterial loads, especially in the culture-positive group and extrapulmonary tuberculosis group. However, its performance in detecting rifampicin resistance is not reliable. Our findings emphasize that Xpert results showing very low bacterial loads should be confirmed when diagnosing rifampin resistance tuberculosis.

What is already known about this topic?:Tuberculosis (TB) is a deadly global infectious disease, and China has a high burden of rifampicin-resistant (RR) and multidrug-resistant (MDR) TB, according to the World Health Organization. Prior treatment, Regional disparities, demographics (age/sex), and comorbidities (e.g., diabetes) are associated with an elevated risk of drug-resistant TB. What is added by this report?:We analyzed 25,978 bacteriologically confirmed pulmonary TB (PTB) cases in 10 sites in Hunan Province and found lower first-line drug resistance rates than national/provincial estimates (2018-2025). Retreatment, age 25-44 years, diabetes, and Lengshuitan residence were independent predictors of resistance. Resistance declined during the pandemic, rebounded slightly thereafter, and displayed a 4.0% annual decrease (statistically significant). There was notable inter-county heterogeneity (intraclass correlation coefficient=0.11). Hunan shifted to molecular testing (82.79% of susceptibility tests in 2021-2025). What are the implications for public health practice?:Surveillance should be intensified for groups at high risk of drug-resistant TB, including retreatment patients, those aged 25-44 years, patients with diabetes and TB, and Lengshuitan residents. A scale-up of molecular diagnostic technologies and increased testing panels to cover isoniazid resistance for accurate resistance assessment should be included. Post-pandemic TB control interventions should continue to optimize the quality of routine surveillance data, and in-depth research on non-laboratory factors that lead to regional disparities in drug resistance rates should be performed.

Transmission of rifampicin-resistant and multidrug-resistant tuberculosis (MDR/RR-TB) is a major driver of the global drug-resistant TB epidemic, yet the contribution of compensatory mutations to its spread remains uncertain. We analyzed 206 non-duplicate MDR/RR-TB isolates collected from five surveillance sites in Hunan Province, China, between 2013 and 2020 using whole-genome sequencing. Transmission clusters were defined by ≤12 single-nucleotide polymorphism (SNP) differences, and compensatory mutations in rpoA, rpoB, and rpoC were identified through a curated mutation catalog and phylogenetic evidence. Clusters were further classified based on whether compensatory mutations predated transmission (C-type), arose after transmission (M-type), or were absent (N-type), with M-type clusters excluded to reduce confounding. Among the 206 isolates, 63 (30.6%) carried at least one compensatory mutation, predominantly in rpoC, and 27 clusters (32.5%) were identified, ranging from 2 to 6 isolates. No significant difference was observed in clustering frequency between compensated and non-compensated strains (21.7% vs 32.8%, P = 0.113), nor in cluster size between C-type and N-type clusters (P = 0.961). These findings demonstrate that compe nsatory mutations do not significantly affect transmission clustering at the population level; instead, prolonged infectious periods associated with diagnostic delays and treatment challenges are more likely to sustain MDR/RR-TB spread, underscoring the importance of rapid detection, timely effective therapy, and robust patient management to curb transmission.IMPORTANCEUnderstanding the drivers of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB) transmission is critical for global TB control. Compensatory mutations in RNA polymerase subunits have been proposed as key contributors to the success of drug-resistant strains, yet population-level evidence has been limited. In this population-based genomic study of 206 MDR/RR-TB isolates collected over 8 years in Hunan Province, China, we systematically examined the association between compensatory mutations and transmission clustering. Our results show that compensatory mutations did not significantly increase clustering frequency or cluster size, indicating a limited impact on the spread of MDR/RR-TB in the community. These findings challenge the prevailing assumption that compensatory evolution is a major determinant of transmission success and provide robust genomic evidence to refine our understanding of drug-resistant TB epidemiology.

This study aimed to identify species identification, molecular typing, and drug resistance characterization of clinically isolated mycobacteria from Anhui, Fujian, and Hunan provinces, and to evaluate the value of whole-genome sequencing (WGS) in predicting drug resistance. WGS and phenotypic drug susceptibility testing (DST) were performed on clinical Mycobacterium isolates from those provinces. Bioinformatics analysis and chi-square test were used to compare inter-provincial differences in drug resistance and lineages. Of the 1,383 isolates, 95.30% belonged to the MTBC and 3.40% were NTM. Lineage 2 (74.07%) and lineage 4 (24.94%) were dominant in MTBC. The overall drug resistance rate was 32.08%, while MDR rate was 11.14%. Hunan had significantly higher MDR and RIF resistance rates than the other provinces (p < 0.05). INH had the highest resistance rate (19.37%). Fluoroquinolones exhibited significant inter-provincial differences in resistance rates. WGS exhibited >85% sensitivity and >96% specificity for first-line drug resistance prediction, and >77% sensitivity and >97% specificity for second-line drug resistance. Additionally, 87.92% of isolates showed consistent genotypic and phenotypic DST results. MTBC isolates exhibited pronounced inter-provincial variation in lineage composition. Geographical differences existed in drug resistance, particularly for MDR and RIF resistance. WGS was reliable for predicting first-line drug resistance, but unknown resistance mechanisms were detected, requiring further exploration to optimize prediction.

IntroductionDigital droplet PCR (ddPCR) offers high sensitivity and absolute quantification without standard curves, making it promising for tuberculosis (TB) detection in paucibacillary samples. This study evaluated the diagnostic performance of a ddPCR assay targeting IS6110 and 23S rRNA for pulmonary tuberculosis (PTB) and tuberculous pleurisy (TP).MethodsA total of 436 patients (245 suspected PTB, 191 suspected TP) were prospectively enrolled. ddPCR was performed on sputum, bronchoalveolar lavage fluid, and pleural effusion samples. Diagnostic performance was compared with Xpert MTB/RIF and culture.ResultsIn suspected PTB, ddPCR showed significantly higher sensitivity than Xpert (49.70% vs. 38.10%, p < 0.001), while Xpert showed higher specificity (98.65% vs. 87.70%). In suspected TP, ddPCR also demonstrated superior sensitivity (17.54% vs. 10.40%, p < 0.001). For NTM detection, ddPCR achieved 60% sensitivity and 99.58% specificity.DiscussionddPCR offers improved sensitivity for PTB and TP diagnosis, particularly in paucibacillary cases, though its lower specificity necessitates confirmatory testing. The dual-target design enables simultaneous MTB/NTM differentiation, providing clinical utility for guiding appropriate therapy.

Discordant drug susceptibility testing (DST) results between the Bactec MGIT 960 system (MGIT) and the GenoType MTBDRplus assay (MTBDRplus) for isoniazid (INH) complicate clinical decision-making. In this study, we performed minimum inhibitory concentration (MIC) assays and whole-genome sequencing (WGS) on 53 Mycobacterium tuberculosis strains identified as INH-resistant by MGIT but INH-susceptible by MTBDRplus. The variants conferring INH resistance were evaluated by the WHO mutation catalogue. Our results showed that only five strains carried variants classified as "associated with resistance" (Group 1/2), including katG Trp39STOP, katG Ser315Asn, inhA -154G>A, and inhA Ser94Ala. In addition, 44 strains carried 70 variants classified as "Group 3: Uncertain significance" across nine genes, including katG, ahpC, inhA, Rv0010c, Rv1129c, Rv2752c, mshA, dnaA, and Rv1258c. The remaining four strains carried no variants (Groups 1-3) linked to INH resistance. No significant difference in the prevalence of high-level INH resistance was observed between lineage 2 and lineage 4 strains (χ² = 0.232, P = 0.630). Our findings indicate that the variants classified as "uncertain significance" may be the main genetic determinants causing discordant results, highlighting their associations with INH resistance that need to be further investigated. IMPORTANCE:This study addresses a critical challenge in drug susceptibility testing (DST): the discrepancies in DST results for isoniazid (INH) between the Bactec MGIT 960 system and the GenoType MTBDRplus assay. These discordant results significantly complicate treatment decisions, potentially leading to suboptimal patient outcomes. Using MIC assays and WGS on 53 clinical Mycobacterium tuberculosis strains, we provide valuable insights into the genetic basis of INH resistance. Our findings showed that only a small fraction of strains carried variants definitively linked to INH resistance, while a larger number harbored variants of uncertain significance across multiple genes, underscoring the complexity of INH resistance mechanisms. This study highlights the urgent need to refine our understanding of these "Group 3: uncertain significance" variants, as they appear to be a primary driver of the discrepancies. Additionally, this study emphasizes the importance of integrating advanced sequencing tools into DST to improve the accuracy of INH resistance detection.

Objective: To evaluate the clinical diagnostic ability of automated simultaneous amplification and testing (AutoSAT) method in suspected pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) by comparing with Xpert MTB/RIF (Xpert) method against the composite reference standard. Method: Patients with suspected PTB or EPTB were enrolled consecutively from two provincial tuberculosis designated hospitals between August 2022 and December 2023. Clinical specimens were collected for routine clinical tests and AutoSAT. Results: The 297 patients including 90 PTB suspects and 207 EPTB suspects were eligible for final analysis. Among the 63 confirmed PTB, the sensitivity of Xpert was significantly higher than that of AutoSAT (30.16% vs. 28.57%, p < 0.001). Of the 18 bacteriologically confirmed PTB, equal sensitivity (94.44%) was observed between Xpert and AutoSAT. The higher specificity of AutoSAT than Xpert (100% vs. 96.30%, p = 0.803) was observed among PTB. The higher sensitivity of AutoSAT than Xpert was observed among confirmed EPTB (22.07% vs. 18.62%, p < 0.001) and bacteriologically confirmed EPTB patients (92.31% vs. 84.62%, p = 0.53), but the specificity (100%) was equal. And AutoSAT yielded higher sensitivity on pleural fluid (14.91% vs. 9.65%, p = 0.276). The same specificities (100%) were observed on different types of specimens. Conclusion: AutoSAT is an accurate, sensitive and rapid method for the detection of Mycobacterium tuberculosis in both PTB and EPTB patients. AutoSAT is highly effective in bacteriologically confirmed PTB, and outperforms Xpert in paucibacillary EPTB patients, especially for pleural fluid specimen detection.

ObjectiveOsteoarticular tuberculosis (OATB) is one of the most common forms of extrapulmonary tuberculosis; however, limited epidemiological data are available on this public health concern worldwide, especially in developing countries. This study aimed to analyze the clinical epidemiology and drug resistance characteristics of OATB cases in Hunan province which located in South-central China.MethodsWe retrospectively enrolled OATB patients with Mycobacterium tuberculosis culture positive at Hunan Chest Hospital from January 2013 through March 31, 2022. The multiple demographic, clinical variables and drug susceptibility data of the patients were collected from the hospital’s electronic patient records. Descriptive statistical methods, Chi-square test and logistic regression analysis were employed as statistical methods.ResultsOf the 269 OATB cases, 197 (73.23%) were males, 206 (76.85%) were farmers; patients’ ages ranged from 5 to 85 years, 57 (21.19%) aged at 20–29 years old and 52 (19.33%) aged at 60–69 years old. In terms of the disease, 177 (65.80%) had spinal TB with most occurrence in lumbar vertebrae (26.02%, 70/269), multiple spinal sites (18.96%, 51/269) and thoracic vertebrae (15.24%, 41/269). Outside of the spine, OATB mainly occurred in the lower limb (13.38%, 36/269). In terms of drug resistance, 40 (14.87%) and 72 (26.77%) were resistant to rifampicin (RFP) and isoniazid (INH) respectively; 38 (14.13%) were multi-drug resistant (MDR), and a total of 78 (29.00%) isolates were drug resistant. OATB patients aged 40–49 years old (compared to those aged ≥70 years) and from the west of Hunan province, China (compared to those from the center of Hunan) were at risk for developing RR/MDR (ORs were 5.057 and 4.942, respectively; 95% CIs were 1.009–25.342 and 1.458–16.750, respectively).ConclusionIn South-central China, OATB mainly affected males, farmers and those aged 20–29 and 60–69 years old. Spinal TB is prone to occur in the lumbar and multiple spinal sites. The resistance situation of OATB was serious, and people aged 40–49 years old and patients from the west of Hunan were risk factors of RR/MDR. All these findings will help to improve the prevention, diagnosis and treatment strategies of OATB.

OBJECTIVES:This study investigated the transmission patterns of tuberculosis (TB) and its associated risk factors in Hunan province to inform the development of prevention and control strategies in the region. METHODS:An 8-year retrospective population-based genomic epidemiological study was conducted. Genomic clusters were defined using distance thresholds of 12-single-nucletide-polymorphisms. Risk factors associated with TB transmission were analyzed using logistic regression model. Kernel Density analysis was used to locate hotspots where transmission occurred. RESULTS:Among 2649 TB cases included in this study, 275 clusters were identified, with an overall clustering rate of 24.7% (654/2649). Nearly 95% (620/654) of clustered strains were isolated from the same county. Of the 275 clusters, 23 (8.4%, 23/275) had differences in drug-resistant profiles, with FQs resistance mutations occurring most frequently (52.2%, 12/23). Multivariate analysis identified male TB patients, those aged 30-60 years, ethnic minorities, nonfarmers, retreated TB patients, and individuals infected with MDR/RR-TB as independent risk factors for TB transmission (P < 0.05). Kernel density analysis showed that among the 5 drug-resistant surveillance sites, Leiyang had the highest clustering rate, followed by Yongshun, Qidong, Hecheng, and Taojiang. CONCLUSION:Recent transmission in the region is predominantly occurring within counties. The risk factors related to TB transmission and the hotspots where transmission occurs can provide a scientific basis for the formulation of targeted TB prevention and control strategies.

Background:China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Methods:Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. Results:The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. Conclusion:DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.

Objectives: This study aimed to investigate the epidemiological and drug resistance (DR) characteristics of extrapulmonary tuberculosis (EPTB) in South-Central China. Methods: EPTB inpatients who were culture-positive for Mycobacterium tuberculosis were retrospectively included in a study at a provincial TB hospital in Hunan, a province in South-Central China, from January 2013 to December 2021. Demographic, clinical, and drug susceptibility data were retrieved from TB treatment records. Descriptive statistical methods and a Chi-squared test were used to analyze the epidemiological and DR characteristics of EPTB patients. A logistic regression model was used to explore the risk factors of rifampicin-resistant/multidrug-resistant (RR/MDR)-EPTB. Results: A total of 1,324 cases were included. The majority of EPTB patients were in the age range of 20-29 years, were predominantly men (male-to-female ratio: 2.03), and were farmers (65.63%). Most EPTB cases were found in 2013 and 2017 from 2013 to 2021. The most prevalent subtypes of EPTB were lymphatic TB (29.83%, 395/1,324), multiple EPTB (20.85%, 276/1,324), and musculoskeletal TB (14.65%, 194/1,324). Musculoskeletal TB and genitourinary TB predominantly presented as exclusive EPTB forms, while lymphatic TB and pharyngeal/laryngeal TB often co-occurred with pulmonary TB (PTB). Drug susceptibility testing results showed that total DR rates (resistance to any of RFP, isoniazid [INH], streptomycin [STR], and/or ethambutol [EMB]) and RR/MDR rates in EPTB were 25.23% and 12.39%, respectively. Musculoskeletal TB exhibited the highest rates of total DR (31.40%), INH resistance (28.90%), STR resistance (20.10%), EMB resistance (6.20%), MDR (13.90%), and poly-DR (6.70%). The multivariable logistic regression model showed that patients aged from 20 to 59 years (compared to those aged 10 years), workers (compared to retirees), and EPTB patients from the south and west of Hunan (compared to those from the east of Hunan) were at an increased risk of developing RR/MDR EPTB (all OR values > 1). Conclusion: Our study provided a detailed account of the epidemiological and DR characteristics of EPTB in Hunan province, China. The significant DR rates, particularly in musculoskeletal TB cases, highlight the need for timely diagnosis, effective drug susceptibility testing, and the development of more effective treatment regimens for EPTB, especially targeting musculoskeletal TB treatments.

ObjectivesTo investigate the clinical epidemiological and drug resistance (DR) characteristics of lymph node tuberculosis (LNTB) in Hunan Province which locates in South-central China, and to provide scientific clues for effective prevention and treatment of LNTB.MethodsWe retrospectively collected LNTB patients with Mycobacterium tuberculosis culture positive at Hunan Chest Hospital, the biggest TB reference hospital in South-central China, from January 2013 to December 2021. The multiple demographic, clinical and drug susceptibility data of patients were collected from the hospital’s electronic patient records. Descriptive statistical methods, Chi-square test and logistic regression analysis were employed as statistical methods.ResultsOf the 577 LNTB cases, 373 (64.64%) were males, 352 (61.01%) were farmers; majority (161, 33.10%) aged at 20–29 years old; 147 (25.48%) had simple LNTB, 350 (60.66%) had LNTB combined with pulmonary TB (PTB) (defined as LNTB-PTB), and 80 (13.86%) had LNTB combined with other extrapulmonary TB (EPTB) (defined as LNTB-EPTB). A total of 345 (59.79%, 345/577) LNTB patients had cervical node infection, and the simple LNTB patients (81.63%, 120/147) had higher proportion of this infection than LNTB-PTB (51.71%, 181/350) and LNTB-EPTB (55.00%, 44/80) (both p values <0.017), respectively. LNTB-EPTB was more inclined to have abdominal tuberculous LNs (20%, 16/80) and at least four tuberculous lesions (22.50%, 18/80) than simple LNTB and LNTB-PTB. Seventy-seven (13.34%) and 119 (20.62%) were resistant to rifampicin (RIF) and isoniazid (INH), respectively; 72 (12.48%) were multi-drug resistant (MDR), and a total of 150 (26.00%) were DR (resistant to at least one of RIF, INH, ethambutol and streptomycin). LNTB patients aged 30–34 and 50–54 years old (compared to those aged <30 years) were independent predictors of RIF resistance (RR) (ORs were 3.47 and 2.83, respectively; 95% CIs were 1.64–7.35 and 1.08–7.46, respectively).ConclusionOur study disclosed the epidemiological and DR characteristics of LNTB in Hunan Province, China. High LNTB prevalence was found in younger people while high RR LNTB prevalence was found in older ones, suggesting that we should conduct further studies to clarify the occurrence of RR in LNTB, meanwhile, strengthen the diagnoses and treatments of LNTB to prevent the emergence of RR.

目的 探讨中性粒细胞分化抗原CD64(nCD64)作为一种新的生物标志物在结核病诊断中的应用价值.方法 选取2019至2020年在湖南省胸科医院就诊的部分肺结核患者和非结核肺部疾病患者,以及健康对照人群为研究对象,收集患者外周血并通过流式细胞术进行nCD64检测,绘制受试者工作曲线(ROC),计算其灵敏度和特异度.同时,以干扰素释放试验(IGRA)为对照,评价其临床诊断参考价值.结果 共纳入141例结核病患者、85例非结核肺部疾病患者(包括非结核分枝杆菌病患者52例和其他呼吸道疾病患者33例)以及76例健康对照组(包括结核潜伏感染者22例和非结核潜伏感染的健康人群54例).结核病患者的nCD64指数为2.03(0.71,5.58),均显著高于非结核肺部疾病患者[1.48(1.14,2.56)](t=3.06,P＜0.01)、结核潜伏感染者[0.57(0.25,0.71)](t=4.312,P＜0.01)和健康人群[0.58(0.33,0.79)](t=4.322,P＜0.01),差异均有统计学意义(P＜0.01).nCD64指数用于结核病诊断的灵敏度、特异度、阳性预测值、阴性预测值和准确度分别为80.9％、72.4％、84.4％、67.1％和77.9％,同IGRA结果相比较,差异均无统计学意义,Kappa值为0.657,两种方法具有较好的一致性.结论 nCD64指数在检测活动性结核病患者中具有良好的灵敏度和特异度,作为一个诊断标志物,在诊断结核病和鉴别结核潜伏感染者和非结核分枝杆菌病中具有临床诊断价值.

BackgroundTuberculosis 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.MethodsA 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.ResultsOf 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.ConclusionEndogenous 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.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

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.

Background: Tuberculosis (TB) seriously threatens individual and public health. Recently, TB outbreaks in schools have been reported more frequently in China and have attracted widespread attention. We reported three TB outbreaks in high schools in Hunan Province, China.Methods: When a tuberculosis patient was reported in a school, we carried out field epidemiological investigations, including tuberculin skin testing (TST), chest X-ray (CXR) and laboratory test for all close contacts, and whole-genome sequencing (WGS) analyses to understand the transmission patterns, the causes and the risk factors for the outbreaks, thereby providing a foundation for the control of TB epidemics in schools.Results: A total of 49 students with TB patients were identified in the three schools where TB outbreaks occurred, including nine patients in School A, 14 patients in School B, and 26 patients in School C. In Schools A, B and C, the putative attack rates in the classes of the index case were 13.8% (8/58), 7.6% (5/66), and 40.4% (21/52), while the putative attack rates of expanding screening in the school were 0.3% (1/361), 0.2% (9/3955), and 0.2% (5/2080), respectively. Thirteen patients had patient delay, with a median delay interval of 69 days (IQR 30.5-113 days). Twelve patients had a healthcare diagnostic delay with a median delay interval of 32 days (IQR 24-82 days). Phylogenetic analysis of culture-positive patients revealed that most of them shared a small genetic distance (<= 12 SNPs), with three separate genetic clusters (including one MDR-TB genomic cluster), indicating the recent transmission of Mycobacterium tuberculosis strains.Conclusion: This combination of field investigation and WGS analysis revealed the transmission of three TB outbreaks in schools. Reinforced implementation is needed to improve timely case finding and reduce diagnosis delay in routine TB control in the school population.

目的 对2019-2020年湖南省分离的非结核分枝杆菌(NTM)进行菌种鉴定,并分析其分布特征,为非结核分枝杆菌病的防治提供基础科学依据.方法 收集2019-2020年湖南省胸科医院疑似结核病患者分离到的分枝杆菌临床分离株,通过MPB64蛋白免疫胶体金法、PNB(对硝基苯甲酸)培养基生长试验进行结核分枝杆菌复合群和NTM鉴定,应用16SrRNA和Hsp65测序分析法对NTM菌株进行菌种鉴定.结果 共收集到6 515份分枝杆菌阳性培养物,初步鉴定为NTM共525株,占比8.06％,其中分离于男性患者285株,占比54.29％;女性患者240株,占比45.71％;人群分布以农民为主,共367株,占比69.90％;年龄分布以40岁以上中老年患者最多,共402株,占比76.57％.2019-2020年NTM菌种分布达27种,菌种分布位列前4者分别为脓肿分枝杆菌(190株,36.19％)、胞内分枝杆菌(174株,33.14％)、鸟分枝杆菌(64株,12.19％)、戈登分枝杆菌(47株,8.95％);其它50株(9.52％)包含23种NTM.脓肿分枝杆菌主要在郴州市、湘西 自治州地区、衡阳市和永州市等地区流行;胞内分枝杆菌主要在郴州市、岳阳市和湘潭市流行.结论 2019-2020年湖南地区分枝杆菌中NTM检出率仍保持较高水平,以脓肿分枝杆菌、胞内分枝杆菌、鸟分枝杆菌和戈登分枝杆菌为主;感染人群以男性、中老年、农民为主.