Analysis of epidemiological characteristics of extrapulmonary tuberculosis from South-Central China.

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.

Different Contributions of embB and ubiA Mutations to Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates.

Objective: To explore the association between the variant mutations within embB and ubiA, and the degree of ethambutol (EMB) resistance of Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods: A total of 146 M. tuberculosis isolates were used to determine the minimum inhibitory concentrations (MICs) of EMB with a 96-well microplate-based assay. The mutations within embB and ubiA among these isolates were identified with DNA sequencing. Moreover, a multivariate regression model and a computer model were established to assess the effects of mutations on EMB resistance. Results: Our data showed that overall 100 isolates exhibited 28 mutated patterns within the sequenced embB and ubiA. Statistical analysis indicated that embB mutations Met306Val, Met306Ile, Gly406Ala, and Gln497Arg, were strongly associated with EMB resistance. Of these mutations, Met306Val and Gln497Arg were significantly associated with high-level EMB resistance. Almost all multiple mutations occurred in high-level EMB-resistant isolates. Although the mutation within ubiA accompanied with embB mutation presented exclusively in EMB-resistant isolates, four single ubiA mutations (Ala39Glu, Ser173Ala, Trp175Cys, and Val283Leu) leading to protein instability were observed in EMB-susceptible isolates. Conclusion: This study highlighted the complexity of EMB resistance. Some individual mutations and multiple mutations within embB and ubiA contributed to the different levels of EMB resistance.

Analysis on epidemiological and drug resistance characteristics of lymph node tuberculosis from Hunan province, China.

Objectives: To 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. Methods: We 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. Results: Of 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). Conclusion: Our 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.

High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Background: Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. Methods: This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Results: Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. Conclusion: This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Recombinant BCG vaccine expressing multistage antigens of Mycobacterium tuberculosis provides long-term immunity against tuberculosis in BALB/c mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) persistently kills nearly 1.5 million lives per year in the world, whereas the only licensed TB vaccine BCG exhibits unsatisfactory efficacy in adults. Taking BCG as a vehicle to express Mtb antigens is a promising way to enhance its efficacy against Mtb infection. In this study, the immune efficacy of recombination BCG (rBCG-ECD003) expressing specific antigens ESAT-6, CFP-10, and nDnaK was evaluated at different time points after immunizing BALB/c mice. The results revealed that rBCG-ECD003 induced multiple Th1 cytokine secretion including IFN-γ, TNF-α, IL-2, and IL-12 when compared to the parental BCG. Under the action of PPD or ECD003, rBCG-ECD003 immunization resulted in a significant increase in the proportion of IL-2+ and IFN-γ+IL-2+ CD4+T cells. Importantly, rBCG-ECD003 induced a stronger long-term humoral immune response without compromising the safety of the parental BCG vaccine. By means of the protective efficacy assay in vitro, rBCG-ECD003 showed a greater capacity to inhibit Mtb growth in the long term. Collectively, these features of rBCG-ECD003 indicate long-term protection and the promising effect of controlling Mtb infection.

Comparison of the Immunogenicity and Efficacy of rBCG-EPCP009, BCG Prime-EPCP009 Booster, and EPCP009 Protein Regimens as Tuberculosis Vaccine Candidates.

Bacillus Calmette-Guérin (BCG) is the only widely used prophylactic tuberculosis (TB) vaccine that can prevent severe TB in infants. However, it provides poor protection in adults, and therefore, there is ongoing research into new TB vaccines and immunization strategies with more durable immune effects. The recombinant BCG and BCG prime-protein booster are two important vaccine strategies that have recently been developed based on BCG and could improve immune responses. In this study, three immune strategies based on four protective antigens, namely, ESAT-6, CFP-10, nPPE18, and nPstS1, were applied to construct recombinant rBCG-EPCP009, EPCP009 subunit protein, and BCG prime-EPCP009 booster vaccine candidates. The short- and long-term immune effects after vaccination in Balb/c mice were evaluated based on humoral immunity, cellular immunity, and the ability of spleen cells to inhibit in vitro mycobacterial growth. At 8 and 12 weeks after the initial immunization, splenocytes from mice inoculated with the BCG prime-EPCP009 protein booster secreted higher levels of PPD- and EPCP009-specific IFN-γ, IL-2, TNF-α, IL-17, GM-CSF, and IL-12 and had a higher IFN-γ+CD4+ TEM:IL-2+CD8+ TCM cell ratio than splenocytes from mice inoculated with the rBCG-EPCP009 and EPCP009 proteins. In addition, the EPCPE009-specific IgG2a/IgG1 ratio was slightly higher in the BCG prime-EPCP009 protein booster group than in the other two groups. The in vitro mycobacterial inhibition assay showed that the splenocytes of mice from the BCG prime-EPCP009 protein booster group exhibited stronger inhibition of Mycobacterium tuberculosis (M. tuberculosis) growth than the splenocytes of mice from the other two groups. These results indicate that the BCG prime-EPCP009 protein booster exhibited superior immunogenicity and M. tuberculosis growth inhibition to the parental BCG, rBCG-EPCP009, and EPCP009 proteins under in vitro conditions. Thus, the BCG prime-EPCP009 protein booster may be important for the development of a more effective adult TB vaccine.

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Introduction: Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. Methods: In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. Results: ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. Conclusion: The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

The acetylation of pknH is linked to the ethambutol resistance of Mycobacterium tuberculosis.

EmbR, a substrate of pknH in Mycobacterium tuberculosis (Mtb), is related to the ethambutol (EMB) resistance. This study aimed to investigate the relationship between acetylation of pknH and the resistance of EMB mono-resistant Mtb. The EMB mono-resistant Mtb strain was constructed based on the MYCOTB and the Löwenstein-Jensen (LJ) proportion method. The growth kinetics was used to evaluate the bacterial growth. Escherichia coli, as the host of Mtb, was used for cloning and protein purification. Moreover, the immunoprecipitation was performed along with western blot to evaluate the EmbR phosphorylation and pknH acetylation. Each independent experiment was conducted in triplicate. EMB mono-resistant Mtb strain was successfully constructed according to the results of MIC values of 14 anti-Mtb drugs. The EMB resistant (ER) Mtb strain showed faster growth than the wild-type (WT) Mtb strain, and the difference was statistically significant. Moreover, pknH robustly phosphorylates EmbR, and pknH and acetylated pknH protein levels were downregulated in ER strain. The acetylation of pknH may reduce the phosphorylation of EmbR to inhibit the growth of Mtb strain. Enhancing the acetylation of pknH may be a promising method to inhibit the EMB resistance against Mtb.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

Association of Single-Nucleotide Polymorphisms in the VDR Gene with Tuberculosis and Infection of Beijing Genotype Mycobacterium tuberculosis.

Background: The aim of the present study was to investigate the association between vitamin D receptor (VDR) gene polymorphism and tuberculosis susceptibility, as well as the potential interaction of host genetic factors with the heterogeneity of Mycobacterium tuberculosis in the population from Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 221 tuberculosis patients as the case group and 363 staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in the VDR were detected by sequencing. M. tuberculosis isolates were collected from the case group and identified as Beijing or non-Beijing lineage by multiplex PCR. Propensity score (PS), univariate analysis and multivariable logistic regression models were used to perform the analysis. Results: Our results showed that the allele and genotype frequencies of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in VDR were not correlated with tuberculosis susceptibility or lineages of M. tuberculosis. Two out of six loci of the VDR gene formed one haplotype block, and none of the haplotypes was found to correlate with tuberculosis susceptibility or lineages of M. tuberculosis infected. Conclusion: Polymorphisms in the VDR gene may not indicate susceptibility to tuberculosis. There was also no evidence on the interaction between the VDR gene of host and the lineages of M. tuberculosis in the population from Xinjiang, China. Further studies are nonetheless required to prove our conclusions.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Genetic Characterization Conferred Co-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Isolates from Southern Xinjiang, China.

Background: Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. Methods: From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Results: Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). Conclusion: This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.

Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates.

Tuberculosis (TB) is the leading cause of death from infectious diseases worldwide, and developing a new TB vaccine is a priority for TB control. Combining multiple immunodominant antigens to form a novel multicomponent vaccine with broad-spectrum antigens to induce protective immune responses is a trend in TB vaccine development. In this study, we used T-cell epitope-rich protein subunits to construct three antigenic combinations: EPC002, ECA006, and EPCP009. Fusion expression of purified protein EPC002f (CFP-10-linker-ESAT-6-linker-nPPE18), ECA006f (CFP-10-linker-ESAT-6-linker-Ag85B), and EPCP009f (CFP-10-linker-ESAT-6-linker-nPPE18-linker-nPstS1) and recombinant purified protein mixtures EPC002m (mix of CFP-10, ESAT-6, and nPPE18), ECA006m (mix of CFP-10, ESAT-6, and Ag85B), and EPCP009m (mix of CFP-10, ESAT-6, nPPE18, and nPstS1) were used as antigens, formulated with alum adjuvant, and the immunogenicity and efficacy were analyzed using immunity experiments with BALB/c mice. All protein-immunized groups elicited higher levels of humoral immunity, including IgG and IgG1. The IgG2a/IgG1 ratio of the EPCP009m-immunized group was the highest, followed by that of the EPCP009f-immunized group, which was significantly higher than the ratios of the other four groups. The multiplex microsphere-based cytokine immunoassay revealed that EPCP009f and EPCP009m induced the production of a wider range of cytokines than EPC002f, EPC002m, ECA006f, and ECA006m, which included Th1-type (IL-2, IFN-γ, TNF-α), Th2-type (IL-4, IL-6, IL-10), Th17-type (IL-17), and other proinflammatory cytokines (GM-CSF, IL-12). The enzyme-linked immunospot assays demonstrated that the EPCP009f- and EPCP009m-immunized groups had significantly higher amounts of IFN-γ than the other four groups. The in vitro mycobacterial growth inhibition assay demonstrated that EPCP009m inhibited Mycobacterium tuberculosis (Mtb) growth most strongly, followed by EPCP009f, which was significantly better than that of the other four vaccine candidates. These results indicated that EPCP009m containing four immunodominant antigens exhibited better immunogenicity and Mtb growth inhibition in vitro and may be a promising candidate vaccine for the control of TB.

Molecular Epidemiology of Clinical Mycobacterium tuberculosis Isolates from Southern Xinjiang, China Using Spoligotyping and 15-Locus MIRU-VNTR Typing.

Background: In the last decades, the molecular epidemiological investigation of Mycobacterium tuberculosis has significantly increased our understanding of tuberculosis epidemiology. However, few such studies have been done in southern Xinjiang, China. We aimed to clarify the molecular epidemic characteristics and their association with drug resistance in the M. tuberculosis isolates circulating in this area. Methods: A total of 347 isolates obtained from southern Xinjiang, China between Sep, 2017 and Sep, 2019 were included to characterize using a 15-locus MIRU-VNTR (VNTR-15China) typing and spoligotyping, and test for drug susceptibility profiles. Then the lineages and clustering of the isolates were analyzed, as well as their association with drug resistance. Results: Spoligotyping results showed that 60 spoligotype international types (SITs) containing 35 predefined SITs and 25 Orphan or New patterns, and 12 definite genotypes were found, and the top three prevalent genotypes were Beijing genotype (207, 59.7%), followed by CAS1-Delhi (46, 13.6%), and Ural-2 (30, 8.6%). The prevalence of Beijing genotype infection in the younger age group (≤30) was more frequent than the two older groups (30~59 and ≥60 years old, both P values <0.05). The Beijing genotype showed significantly higher prevalence of resistance to isoniazid, rifampicin, ethambutol, multi-drug or at least one drug than the non-Beijing genotype (All P values ≤0.05). The estimated proportion of tuberculosis cases due to transmission was 18.4% according to the cluster rate acquired by VNTR-15China typing, and the Beijing genotype was the risk factor for the clustering (OR 9.15, 95% CI: 4.18-20.05). Conclusion: Our data demonstrated that the Beijing genotype is the dominant lineage, associated with drug resistance, and was more likely to infect young people and contributed to tuberculosis transmission in southern Xinjiang, China. These findings will contribute to a better understanding of tuberculosis epidemiology in this area.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice.

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

rpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis.

Objective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.

Association of Mannose-Binding Lectin 2 Gene Polymorphism with Tuberculosis Based on Mycobacterium tuberculosis Lineages.

Purpose: Polymorphisms in MBL2 may contribute to the susceptibility to tuberculosis. The aim of the present study was to determine the associations of the polymorphisms of five loci (rs1800450, rs1800451, rs7096206, rs7095891, and rs11003125) in the MBL2 gene with susceptibility to tuberculosis and specific lineages of Mycobacterium tuberculosis causing tuberculosis in the Uyghur population of Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 170 Uyghur tuberculosis patients as the case group and 147 Uyghur staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of five loci in MBL2 of human were detected by sequencing. Whole-genome sequencing was applied in 68 M. tuberculosis isolates from the case group and the data were used to perform genealogy analysis. Results: The distributions of allele and genotype frequencies of five loci in MBL2 varied little between the case and control groups and varied little among the groups, including those infected with different lineages of M. tuberculosis and the control (except those of rs11003125), the P values were all >0.05. The distribution of alleles of rs11003125 was statistically different between patients infected with lineages 3 and 4 M. tuberculosis (χ 2=7.037, P=0.008). The C allele and CC genotype of rs11003125 were found to be protective factors against lineage 4 infection when compared to lineage 3 (ORs were 0.190 and 0.158, respectively; 95% confidence intervals were 0.053~0.690 and 0.025~0.999, respectively). Conclusion: Our results suggested that human's susceptibility to tuberculosis is affected both by the host genetic polymorphisms and the lineage of the M. tuberculosis that people were exposed to. However, due to the limitation of the sample size in the present study, larger sample size and more rigorous design should be guaranteed in future studies.

rpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

OBJECTIVE: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure. METHODS: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed. RESULTS: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance. CONCLUSION: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

Prevalence and Molecular Characteristics Based on Whole Genome Sequencing of Mycobacterium tuberculosis Resistant to Four Anti-Tuberculosis Drugs from Southern Xinjiang, China.

OBJECTIVE: Drug-resistant tuberculosis is a major public health problem, especially in the southern region of Xinjiang, China; however, there is little information regarding drug resistance profiles and mechanism of Mycobacterium tuberculosis in this area. The aim of this study was to determine the prevalence and molecular characteristics of M. tuberculosis resistant to four anti-tuberculosis drugs from this area. METHODS: Three hundred and forty-six isolates from the southern region of Xinjiang, China were included and used to perform phenotypic drug susceptibility testing and whole genome sequencing (WGS). Mutations in seven loci associated with drug resistance, including rpoB for rifampicin (RMP), katG, inhA promoter and oxyR-ahpC for isoniazid (INH), rrs 530 and 912 loops and rpsL for streptomycin (STR), and embB for ethambutol (EMB), were characterized. RESULTS: Among 346 isolates, 106, 60, 70 and 29 were resistant to INH, RMP, STR and EMB, respectively; 132 were resistant to at least one of the four anti-tuberculosis drugs and 51 were multi-drug resistant (MDR). Beijing genotype and retreated patients showed a significantly increased risk for developing MDR tuberculosis. Compared with the phenotypic data, the sensitivity and specificity for WGS to predict resistance were 96.7% and 98.6% for RMP, 75.5% and 97.1% for INH, 68.6% and 99.6% for STR, 93.1% and 93.7% for EMB, respectively. The most common mutations conferring RMP, INH, STR and EMB resistance were Ser450Leu (51.7%) in rpoB, Ser315Thr (44.3%) in katG, Lys43Arg (35.7%) in rpsL and Met306Val (24.1%) in embB. CONCLUSION: This study provides the first information on the prevalence and molecular characters of drug resistant M. tuberculosis in the southern region of Xinjiang, China, which will be helpful for choosing early detection methods for drug resistance (ig, molecular methods) and subsequently initiation of proper therapy of tuberculosis in this area.