Trends of Drug-Resistant Tuberculosis in an Urban and a Rural Area in China: A 10-Year Population-Based Molecular Epidemiological Study.

Objective: Drug resistance is the critical determinant for appropriate tuberculosis (TB) treatment regimens and an important indicator of the local TB burden. We aimed to investigate and compare trends in TB drug resistance in the urban Songjiang District of Shanghai from 2011 to 2020, and the rural Wusheng County of Sichuan Province from 2009 to 2020, to assess the effectiveness of local TB control and treatment programs. Methods: Whole-genome sequencing data of Mycobacterium tuberculosis were used to predict drug-resistance profiles and identify genomic clusters. Clustered, retreated cases of drug-resistant TB with identical resistance mutations, as well as all new resistant cases, were defined as transmitted resistance. The Cochran-Armitage trend test was used to identify trends in the proportions. Differences between groups were tested using the Wilcoxon rank sum or chi-square tests. Results: The annual proportions of rifampicin-resistant (RR), isoniazid-resistant (INH-R) and multidrug-resistant (MDR) TB cases did not change significantly in Songjiang. In Wusheng, however, the percentage of total TB cases that were RR decreased from 13.2% in 2009 to 3.7% in 2020, the INH-R cases decreased from 16.5% to 7.3%, and the MDR cases decreased from 10.7% to 3.7%. In retreated cases, the percentage of drug resistance decreased in both Songjiang and Wusheng, suggesting improved treatment programs. Transmitted resistance accounted for more than two thirds of drug-resistant cases over the entire study periods, and in recent years this proportion has increased significantly in Songjiang. Conclusion: In both urban Songjiang and rural Wusheng, drug-resistant TB is mostly the result of transmission of drug resistant strains and the percentage of transmitted resistance will likely increase with on-going improvements in the TB treatment programs. Reducing the prevalence of drug resistance depends principally upon decreasing transmission through the prompt diagnosis and effective treatment of drug-resistant TB cases.

Performance evaluation of core genome multilocus sequence typing for genotyping of Mycobacterium tuberculosis strains in China: based on multicenter, population-based collection.

PURPOSE: To evaluate the performance of core genome multilocus sequence typing (cgMLST) for genotyping Mycobacterium tuberculosis (M.tuberculosis) Strains in regions where the lineage 2 strains predominate. METHODS: We compared clustering by whole-genome SNP typing with cgMLST clustering in the analysis of WGS data of 6240 strains from five regions of China. Using both the receiver operating characteristic (ROC) curve and epidemiological investigation to determine the optimal threshold for defining genomic clustering by cgMLST. The performance of cgMLST was evaluated by quantifying the sensitivity, specificity and concordance of clustering between two methods. Logistic regression was used to gauge the impact of strain genetic diversity and lineage on cgMLST clustering. RESULTS: The optimal threshold for cgMLST to define genomic clustering was determined to be ≤ 10 allelic differences between strains. The overall sensitivity and specificity of cgMLST averaged 99.6% and 96.3%, respectively; the concordance of clustering between two methods averaged 97.1%. Concordance was significantly correlated with strain genetic diversity and was 3.99 times (95% CI, 2.94-5.42) higher in regions with high genetic diversity (π > 1.55 × 10-4) compared to regions with low genetic diversity. The difference missed statistical significance, while concordance for lineage 2 strains (96.8%) was less than that for lineage 4 strains (98.3%). CONCLUSION : cgMLST showed a discriminatory power comparable to whole-genome SNP typing and could be used to genotype clinical M.tuberculosis strains in different regions of China. The discriminative power of cgMLST was significantly correlated with strain genetic diversity and was slightly lower with strains from regions with low genetic diversity.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

Implications for TB control among migrants in large cities in China: A prospective population-based genomic epidemiology study in Shenzhen.

Internal migrants are a challenge for TB control in large Chinese cities and understanding this epidemiology is crucial for designing effective control and prevention strategies. We conducted a prospective genomic epidemiological study of culture-positive TB patients diagnosed between June 1, 2018 and May 31, 2021 in the Longhua District of Shenzhen. Treatment status was obtained from local and national TB registries and all isolates were sequenced. Genomic clusters were defined as strains differing by ≤12 SNPs. Risk factors for clustering were identified with multivariable analysis and then Bayesian models and TransPhylo were used to infer the timing of transmission within clusters. Of the 2277 culture-positive patients, 70.1% (1596/2277) were migrants: 72.1% (1043/1446) of the migrants patients developed TB within two years of arriving in Longhua; 38.8% within 6 months of arriving; and 12.3% (104/843) had TB symptoms when they arrived. Only 15.4% of Longhua strains were in genomic clusters. More than one third (33.6%) of patients were not treated in Shenzhen but were involved in nearly one third of the recent transmission events. Clustering was associated with migrants not treated in Shenzhen, males, and teachers/trainers. TB in Longhua is prinicipally due to reactivation of infections in migrants, but a proportion may have had clinical or incipient TB upon arrival in the district. Patients diagnosed but not treated in Longhua were involved in recent local TB transmission. Controlling TB in Shenzhen will require strategies to comprehensively diagnose and treat active TB in the internal migrant population.

Genotypic and spatial analysis of transmission dynamics of tuberculosis in Shanghai, China: a 10-year prospective population-based surveillance study.

Background: With improved tuberculosis (TB) control programs, the incidence of TB in China declined dramatically over the past few decades, but recently the rate of decrease has slowed, especially in large cities such as Shanghai. To help formulate strategies to further reduce TB incidence, we performed a 10-year study in Songjiang, a district of Shanghai, to delineate the characteristics, transmission patterns, and dynamic changes of the local TB burden. Methods: We conducted a population-based study of culture-positive pulmonary TB patients diagnosed in Songjiang during 2011-2020. Genomic clusters were defined with a threshold distance of 12-single-nucleotide-polymorphisms based on whole-genome sequencing, and risk factors for clustering were identified by logistic regression. Transmission inference was performed using phybreak. The distances between the residences of patients were compared to the genomic distances of their isolates. Spatial patient hotspots were defined with kernel density estimation. Findings: Of 2212 enrolled patients, 74.7% (1652/2212) were internal migrants. The clustering rate (25.2%, 558/2212) and spatial concentrations of clustered and unclustered patients were unchanged over the study period. Migrants had significantly higher TB rates but less clustering than residents. Clustering was highest in male migrants, younger patients and both residents and migrants employed in physical labor. Only 22.1% of transmission events occurred between residents and migrants, with residents more likely to transmit to migrants. The clustering risk decreased rapidly with increasing distances between patient residences, but more than half of clustered patient pairs lived ≥5 km apart. Epidemiologic links were identified for only 15.6% of clustered patients, mostly in close contacts. Interpretation: Although some of the TB in Songjiang's migrant population is caused by strains brought by infected migrants, local, recent transmission is an important driver of the TB burden. These results suggest that further reductions in TB incidence require novel strategies to detect TB early and interrupt urban transmission. Funding: Shanghai Municipal Science and Technology Major Project (ZD2021CY001), National Natural Science Foundation of China (82272376), National Research Council of Science and Technology Major Project of China (2017ZX10201302-006).

Evaluation of the Cepheid 3-gene host response blood test for tuberculosis diagnosis and treatment response monitoring in a primary-level clinic in rural China.

A rapid, accurate, non-sputum-based triage test for diagnosing tuberculosis (TB) is a high-priority need. Cepheid developed a novel prototype blood test, Xpert Mycobacterium tuberculosis Host Response (Xpert-MTB-HR), which generates a TB score based on the mRNA expression of three genes. We conducted a case-control study with prospective recruitment to evaluate its accuracy in the clinic of the Wusheng County Centers for Disease Prevention and Control in China. We enrolled 149 TB patients, 248 other respiratory diseases (ORD) patients, and 193 healthy controls. In addition, whole-blood samples taken from TB patients after 2, 5, and 6 months of treatment were tested with Xpert-MTB-HR to evaluate its ability to monitor treatment response. Xpert-MTB-HR discriminated between TB and healthy controls with an area under the curve (AUC) of 0.912 (95% CI, 0.878-0.945). With the specificity of 70% envisioned for a triage test, its sensitivity was 90.1% (84.9%-94.6%). Xpert-MTB-HR discriminated between TB and ORD with an AUC of 0.798 (0.750-0.847), and at specificity of 70%, the sensitivity was only 75.8% (68.5%-82.8%). In patients determined by Ultra to have medium or high sputum bacillary loads, with specificity of 70%, the sensitivity for discriminating patients with TB from healthy controls was 100.0% (100.0-100.0) and from patients with ORD, 95.1% (89.8-100.0). The TB scores generally increased by 2 months of treatment and then remained stable. Xpert-MTB-HR met the criteria for a triage test to discriminate between TB and healthy controls, but not between TB and ORD, except when limited to patients with high sputum bacillary loads. Xpert-MTB-HR showed promise for monitoring response to treatment but needs to be further evaluated in larger prospective studies.

Comparison of Xpert MTB/RIF Ultra with Xpert MTB/RIF for the detection of Mycobacterium tuberculosis and rifampicin resistance in a primary-level clinic in rural China.

The Xpert MTB/RIF Ultra (Ultra) is not yet used for the diagnosis of tuberculosis (TB) in China. We compared the performance of the Xpert and Ultra for detecting Mycobacterium tuberculosis and rifampicin resistance in a primary-level clinic in rural China. Sputum samples from suspected pulmonary TB patients were collected and subjected to smear microscopy, liquid culture, Xpert and Ultra tests. We then compared the sensitivity and specificity of Xpert and Ultra for diagnosing TB against liquid culture. Whole-genome sequencing was performed to predict rifampicin resistance and the results were compared with the Xpert and Ultra tests. The sensitivities of Xpert and Ultra were 88.1% and 95.1%, and the specificities were 91.9% and 84.4%, respectively. Among the 61 smear-negative culture-positive patients, the sensitivities of Xpert and Ultra were 80.3% and 91.8%. All Xpert-positive patients were Ultra-positive. Among culture-negative Xpert or Ultra-positive patients, 69.6% were taking anti-TB drugs or had a previous history of TB. Of the samples that Ultra classified as trace, nearly 25% were probably false-positives. Both Xpert and Ultra accurately detected all rifampicin-resistant patients. In conclusion, Ultra was more sensitive than Xpert, especially for smear-negative patients but had decreased specificity with more false-positives, especially with Ultra trace results.

Association of PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms with pulmonary tuberculosis susceptibility in a Chinese population.

BACKGROUND: Autophagy can inhibit the survival of intracellular microorganisms including Mycobacterium tuberculosis (Mtb), and the PI3K/AKT/mTOR pathway plays a crucial role. This study investigated the association between PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms and pulmonary tuberculosis (PTB) susceptibility. METHODS: KEGG pathway and gene ontology (GO) databases were searched for genes belonging to the PI3K/AKT/mTOR and autophagy pathways. Thirty SNPs in nine genes were identified and tested for their associations with tuberculosis in 130 patients with PTB and 271 controls. We constructed genetic risk scores (GRSs) and divided the participants into 3 subgroups based on their GRSs:0-5, 6-10, and 11-16. RESULTS: This analysis revealed that the AKT1 (rs12432802), RPTOR (rs11654508, rs12602885, rs2090204, rs2589144, and rs2672897), and TSC2 (rs2074969) polymorphisms were significantly associated with PTB risk. A decreasing trend was observed (P trend 0.020), in which a lower GRS was associated with a higher risk of PTB ([6-10] vs. [0-5]: OR (95%CI) 0.590 (0.374-0.931); [11-16] vs. [0-5]: OR (95%CI) 0.381 (0.160-0.906)). CONCLUSIONS: Polymorphisms in AKT1, RPTOR, and TSC2 may influence susceptibility to PTB.

The Global Success of Mycobacterium tuberculosis Modern Beijing Family Is Driven by a Few Recently Emerged Strains.

Strains of the Mycobacterium tuberculosis complex (MTBC) Beijing family aroused concern because they were often found in clusters and appeared to be exceptionally transmissible. However, it was later found that strains of the Beijing family were heterogeneous, and the transmission advantage was restricted to sublineage L2.3 or modern Beijing. In this study, we analyzed the previously published genome sequences of 7,896 L2.3 strains from 51 different countries. Using BEAST software to approximate the temporal emergence of L2.3, our calculations suggest that L2.3 initially emerged in northern East Asia during the early 15th century and subsequently diverged into six phylogenetic clades, identified as L2.3.1 through L2.3.6. Using terminal branch length and genomic clustering as proxies for transmissibility, we found that the six clades displayed distinct population dynamics, with the three recently emerged clades (L2.3.4 to L2.3.6) exhibiting significantly higher transmissibility than the older three clades (L2.3.1 to L2.3.3). Of the Beijing family strains isolated outside East Asia, 83.1% belonged to the clades L2.3.4 to L2.3.6, which were also associated with more cross-border transmission. This work reveals the heterogeneity in sublineage L2.3 and demonstrates that the global success of Beijing family strains is driven by the three recently emerged L2.3 clades. IMPORTANCE The recent population dynamics of the global tuberculosis epidemic are heavily shaped by Mycobacterium tuberculosis complex (MTBC) strains with enhanced transmissibility. The infamous Beijing family strain stands out because it has rapidly spread throughout the world. Identifying the strains responsible for the global expansion and tracing their evolution should help to understand the nature of high transmissibility and develop effective strategies to control transmission. In this study, we found that the L2.3 sublineage diversified into six phylogenetic clades (L2.3.1 to L2.3.6) with various transmission characteristics. Clades L2.3.4 to L2.3.6 exhibited significantly higher transmissibility than clades L2.3.1 to L2.3.3, which helps explain why more than 80% of Beijing family strains collected outside East Asia belong to these three clades. We conclude that the global success of L2.3 was not caused by the entire L2.3 sublineage but rather was due to the rapid expansion of L2.3.4 to L2.3.6. Tracking the transmission of L2.3.4 to L2.3.6 strains can help to formulate targeted TB prevention and control.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

The mutational signatures of poor treatment outcomes on the drug-susceptible Mycobacterium tuberculosis genome.

Drug resistance is a known risk factor for poor tuberculosis (TB) treatment outcomes, but the contribution of other bacterial factors to poor outcomes in drug-susceptible TB is less well understood. Here, we generate a population-based dataset of drug-susceptible Mycobacterium tuberculosis (MTB) isolates from China to identify factors associated with poor treatment outcomes. We analyzed whole-genome sequencing (WGS) data of MTB strains from 3196 patients, including 3105 patients with good and 91 patients with poor treatment outcomes, and linked genomes to patient epidemiological data. A genome-wide association study (GWAS) was performed to identify bacterial genomic variants associated with poor outcomes. Risk factors identified by logistic regression analysis were used in clinical models to predict treatment outcomes. GWAS identified fourteen MTB fixed mutations associated with poor treatment outcomes, but only 24.2% (22/91) of strains from patients with poor outcomes carried at least one of these mutations. Isolates from patients with poor outcomes showed a higher ratio of reactive oxygen species (ROS)-associated mutations compared to isolates from patients with good outcomes (26.3% vs 22.9%, t-test, p=0.027). Patient age, sex, and duration of diagnostic delay were also independently associated with poor outcomes. Bacterial factors alone had poor power to predict poor outcomes with an AUC of 0.58. The AUC with host factors alone was 0.70, but increased significantly to 0.74 (DeLong's test, p=0.01) when bacterial factors were also included. In conclusion, although we identified MTB genomic mutations that are significantly associated with poor treatment outcomes in drug-susceptible TB cases, their effects appear to be limited.

Whole-Genome Sequencing Exhibits Better Diagnostic Performance than Variable-Number Tandem Repeats for Identifying Mixed Infections of Mycobacterium tuberculosis.

Mixed infections of Mycobacterium tuberculosis, defined as the coexistence of multiple genetically distinct strains within a single host, have been associated with unfavorable treatment outcomes. Different methods have been used to detect mixed infections, but their performances have not been carefully evaluated. To compare the sensitivity of whole-genome sequencing (WGS) and variable-number tandem repeats (VNTR) typing to detect mixed infections, we prepared 10 artificial samples composed of DNA mixtures from two strains in different proportions and retrospectively collected 1,084 clinical isolates. The limit of detection (LOD) for the presence of a minor strain was 5% for both WGS and VNTR typing. The overall clinical detection rate of mixed infections was 3.7% (40/1,084) for the two methods combined, WGS identified 37/1,084 (3.4%), and VNTR typing identified 14/1,084 (1.3%), including 11 also identified by WGS. Multivariate analysis demonstrated that retreatment patients had a 2.7 times (95% confidence interval [CI], 1.2 to 6.0) higher risk of mixed infections than new cases. Collectively, WGS is a more reliable tool to identify mixed infections than VNTR typing, and mixed infections are more common in retreated patients. IMPORTANCE Mixed infections of M. tuberculosis have the potential to render treatment regimens ineffective and affect the transmission dynamics of the disease. VNTR typing, currently the most widely used method for the detection of mixed infections, detects mixed infections only by interrogating a small fraction of the M. tuberculosis genome, which necessarily limits sensitivity. With the introduction of WGS, it became possible to study the entire genome, but no quantitative comparison has yet been undertaken. Our systematic comparison of the ability of WGS and VNTR typing to detect mixed infections, using both artificial samples and clinical isolates, revealed the superior performance of WGS at a high sequencing depth (~100×) and found that mixed infections are more common in patients being retreated for tuberculosis (TB) in the populations studied. This provides valuable information for the application of WGS in the detection of mixed infections and the implications of mixed infections for tuberculosis control.

Discrepancy in the transmissibility of multidrug-resistant mycobacterium tuberculosis in urban and rural areas in China.

The fitness of multidrug-resistant tuberculosis (MDR-TB) is thought to be an important determinant of a strain's ability to be transmitted. Studies in the laboratory have demonstrated that MDR-TB strains have reduced fitness but the relative transmissibility of MDR-TB versus drug-susceptible (DS) TB strains in human populations remains unresolved. We used data on genomic clustering from our previous molecular epidemiological study in Songjiang (2011-2020) and Wusheng (2009-2020), China, to compare the relative transmissibility of MDR-TB versus DS-TB. Genomic clusters were defined with a threshold distance of 12-single-nucleotide-polymorphisms and the risk for MDR-TB clustering was analyzed by logistic regression. In total, 2212 culture-positive pulmonary TB patients were enrolled in Songjiang and 1289 in Wusheng. The clustering rates of MDR-TB and DS-TB strains were 19.4% (20/103) and 26.3% (509/1936), respectively in Songjiang, and 43.9% (29/66) and 26.0% (293/1128) in Wusheng. The risk of MDR-TB clustering was 2.34 (95% CI 1.38-3.94) times higher than DS-TB clustering in Wusheng and 0.64 (95% CI 0.38-1.06) times lower in Songjiang. Neither lineage 2, compensatory mutations nor rpoB S450L were significantly associated with MDR-TB transmission, and katG S315 T increased MDR-TB transmission only in Wusheng (OR 5.28, 95% CI 1.42-19.21). MDR-TB was not more transmissible than DS-TB in either Songjiang or Wusheng. It appears that the different transmissibility of MDR-TB in Songjiang and Wusheng is likely due to differences in the quality of the local TB control programmes. Suggesting that the most effective way to control MDR-TB is by improving local TB control programmes.

The Evolution and Transmission Dynamics of Multidrug-Resistant Tuberculosis in an Isolated High-Plateau Population of Tibet, China.

On the Tibetan Plateau, most tuberculosis is caused by indigenous Mycobacterium tuberculosis strains with a monophyletic structure and high-level drug resistance. This study investigated the emergence, evolution, and transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Tibet. The whole-genome sequences of 576 clinical strains from Tibet were analyzed with the TB-profiler tool to identify drug-resistance mutations. The evolution of the drug resistance was then inferred based on maximum-likelihood phylogeny and dated trees that traced the serial acquisition of mutations conferring resistance to different drugs. Among the 576 clinical M. tuberculosis strains, 346 (60.1%) carried at least 1 resistance-conferring mutation and 231 (40.1%) were MDR-TB. Using a pairwise distance of 50 single nucleotide polymorphisms (SNPs), most strains (89.9%, 518/576) were phylogenetically separated into 50 long-term transmission clusters. Eleven large drug-resistant clusters contained 76.1% (176/231) of the local multidrug-resistant strains. A total of 85.2% of the isoniazid-resistant strains were highly transmitted with an average of 6.6 cases per cluster, of which most shared the mutation KatG Ser315Thr. A lower proportion (71.6%) of multidrug-resistant strains were transmitted, with an average cluster size of 2.9 cases. The isoniazid-resistant clusters appear to have undergone substantial bacterial population growth in the 1970s to 1990s and then subsequently accumulated multiple rifampicin-resistance mutations and caused the current local MDR-TB burden. These findings highlight the importance of detecting and curing isoniazid-resistant strains to prevent the emergence of endemic MDR-TB. IMPORTANCE Emerging isoniazid resistance in the 1970s allowed M. tuberculosis strains to spread and form into large multidrug-resistant tuberculosis clusters in the isolated plateau of Tibet, China. The epidemic was driven by the high risk of transmission as well as the potential of acquiring further drug resistance from isoniazid-resistant strains. Eleven large drug-resistant clusters consisted of the majority of local multidrug-resistant cases. Among the clusters, isoniazid resistance overwhelmingly evolved before all the other resistance types. A large bacterial population growth of isoniazid-resistant clusters occurred between 1970s and 1990s, which subsequently accumulated rifampicin-resistance-conferring mutations in parallel and accounted for the local multidrug-resistant tuberculosis burden. The results of our study indicate that it may be possible to restrict MDR-TB evolution and dissemination by prioritizing screening for isoniazid (INH)-resistant TB strains before they become MDR-TB and by adopting measures that can limit their transmission.

Association of PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms with pulmonary tuberculosis susceptibility in a Chinese population

ABSTRACT Background: Autophagy can inhibit the survival of intracellular microorganisms including Mycobacterium tuberculosis (Mtb), and the PI3K/AKT/mTOR pathway plays a crucial role. This study investigated the association between PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms and pulmonary tuberculosis (PTB) susceptibility. Methods: KEGG pathway and gene ontology (GO) databases were searched for genes belonging to the PI3K/AKT/mTOR and autophagy pathways. Thirty SNPs in nine genes were identified and tested for their associations with tuberculosis in 130 patients with PTB and 271 controls. We constructed genetic risk scores (GRSs) and divided the participants into 3 subgroups based on their GRSs:0-5, 6-10, and 11-16. Results: This analysis revealed that the AKT1 (rs12432802), RPTOR (rs11654508, rs12602885, rs2090204, rs2589144, and rs2672897), and TSC2 (rs2074969) polymorphisms were significantly associated with PTB risk. A decreasing trend was observed (P trend 0.020), in which a lower GRS was associated with a higher risk of PTB ([6-10] vs. [0-5]: OR (95%CI) 0.590 (0.374-0.931); [11-16] vs. [0-5]: OR (95%CI) 0.381 (0.160-0.906)). Conclusions: Polymorphisms in AKT1, RPTOR, and TSC2 may influence susceptibility to PTB.

Polymorphisms of the BCL2 gene associated with susceptibility to tuberculosis.

Although tuberculosis (TB) is a serious public health concern, we still don't understand why only 10% of people infected will develop the disease. Apoptosis plays a role in the interaction of Mycobacterium tuberculosis (Mtb) with the human host and it may be modified by subtle alterations in the B-cell lymphoma 2 (BCL2) gene, an anti-apoptotic regulatory element. Therefore, we investigated whether there is an association between BCL2 polymorphisms and susceptibility to TB by analyzing 130 TB cases, 108 subjects with latent TB infection (LTBI), and 163 healthy controls (HC). Logistic regression was used to calculate odds ratios (ORs) and 95% confidential intervals (95% CIs) for possible associations between single nucleotide polymorphisms (SNPs) in BCL2 and the risk of tuberculosis. We found that the G allele of rs80030866 (OR=0.62, 95%CI:0.42-0.91, P=0.015), and also the G allele of rs9955190 (OR=0.58, 95%CI:0.38-0.88, P=0.011) were less frequent in the TB group compared with the LTBI group. In addition, individuals with rs2551402 CC genotype were more likely to have LTBI than those with AA genotype (OR=2.166, 95%CI:1.046-4.484, P=0.037). Our study suggests that BCL2 gene polymorphisms may be correlated with susceptibility to both TB and LTBI.

High proportion of tuberculosis transmission among social contacts in rural China: a 12-year prospective population-based genomic epidemiological study.

ABSTRACTTuberculosis (TB) is more prevalent in rural than urban areas in China, and delineating TB transmission patterns in rural populations could improve TB control. We conducted a prospective population-based study of culture-positive pulmonary TB patients diagnosed between July 1, 2009 and December 31, 2020 in two rural counties in China. Genomic clusters were defined with a threshold distance of 12-single-nucleotide-polymorphisms, based on whole-genome sequencing. Risk factors for clustering were identified by logistic regression. Transmission links were sought through epidemiological investigation of genomic-clustered patients. Of 1517 and 751 culture-positive pulmonary TB patients in Wusheng and Wuchang counties, respectively, 1289 and 699 strains were sequenced. Overall, 624 (31.4%, 624/1988) patients were grouped into 225 genomic clusters. Epidemiological links were confirmed in 41.8% (196/469) of clustered isolates, including family (32.7%, 64/196) and social contacts (67.3%, 132/196). Social contacts were generally with relatives, within the community or in shared aggregated settings outside the community, but the proportion of clustered contacts in each category differed between the two sites. The time interval between diagnosis of student cases and contacts was significantly shorter than family and social contacts, probably due to enhanced student contact screening. Transmission of multidrug-resistant (MDR) strains was likely responsible for 81.4% (83/102) of MDR-TB cases, with minimal acquisition of additional resistance mutations. A large proportion of TB transmission in rural China occurred among social contacts, suggesting that active screening and aggressive contact tracing could benefit TB control, but contact screening should be tailored to local patterns of social interactions.

Negative Regulator Nlrc3-like Maintain the Balanced Innate Immune Response During Mycobacterial Infection in Zebrafish.

The NOD-like receptors (NLRs) have been shown to be involved in infection and autoinflammatory disease. Previously, we identified a zebrafish NLR, nlrc3-like, required for macrophage homeostasis in the brain under physiological conditions. Here, we found that a deficiency of nlrc3-like leads to decreased bacterial burden at a very early stage of Mycobacterium marinum infection, along with increased production of pro-inflammatory cytokines, such as il-1ß and tnf-α. Interestingly, myeloid-lineage specific overexpression of nlrc3-like achieved the opposite effects, suggesting that the impact of nlrc3-like on the host anti-mycobacterial response is mainly due to its expression in the innate immune system. Fluorescence-activated cell sorting (FACS) and subsequent gene expression analysis demonstrated that inflammasome activation-related genes were upregulated in the infected macrophages of nlrc3-like deficient embryos. By disrupting asc, encoding apoptosis-associated speck-like protein containing a CARD, a key component for inflammasome activation, the bacterial burden increased in asc and nlrc3-like double deficient embryos compared with nlrc3-like single deficient embryos, implying the involvement of inflammasome activation in infection control. We also found extensive neutrophil infiltration in the nlrc3-like deficient larvae during infection, which was associated with comparable bacterial burden but increased tissue damage and death at a later stage that could be alleviated by administration of dexamethasone. Our findings uncovered an important role of nlrc3-like in the negative regulation of macrophage inflammasome activation and neutrophil infiltration during mycobacterial infection. This highlights the importance of a balanced innate immune response during mycobacterial infection and provides a potential molecular basis to explain how anti-inflammatory drugs can improve treatment outcomes in TB patients whose infection is accompanied by a hyperinflammatory response.

Comprehensive genomic analysis of Mycobacterium tuberculosis reveals limited impact of high-fitness genotypes on MDR-TB transmission.

OBJECTIVES: Environmental and host-related factors that contribute to the transmission of multidrug-resistant tuberculosis (MDR-TB) have become an increasing concern, but the impact of bacterial genetic factors associated with bacterial fitness on MDR-TB transmission is poorly understood. Here, we present a global view of the correlation between common fitness-related genotypes and MDR-TB transmission by analyzing a representative number of MDR-TB isolates. METHODS: We assembled a global whole genome sequencing (WGS) dataset of MDR-TB strains collected through retrospective cohorts or population-based approaches using public databases and literature curation. WGS-based clusters were defined as groups of strains with genomic difference of ≤ 5 SNPs. RESULTS: We curated high-quality WGS data of 4696 MDR-TB isolates from 17 countries with a mean clustering rate of 48% (range 0-100%). Correlational analysis showed that increased risk of MDR-TB strain clustering was not associated with compensatory mutations (OR 1.07, 95% CI 0.72-1.59), low-fitness cost drug-resistant mutations (katG S315T: OR 1.42, 95% CI 0.82-2.47; rpoB S450L: OR 1.26, 95% CI 0.87-1.83) or Lineage 2 (OR 1.50, 95% CI 0.95-2.39). CONCLUSIONS: The factors most commonly thought to increase bacterial fitness were not significantly associated with increased MDR-TB transmission, and thus do not appear to be major contributors to the current epidemic of MDR-TB.