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Weicong Ren,1,* Haoran Li,1,* Can Guo,1,2 Yuanyuan Shang,1 Wei Wang,1 Xuxia Zhang,1 Shanshan Li,1 Yu Pang1 1Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, People’s Republic of China; 2Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yu Pang; Shanshan Li, Email pangyupound@163.com; lss9011@126.comBackground: Aspergillus fumigatus-induced chronic pulmonary aspergillosis (CPA), the most common pulmonary tuberculosis (TB) sequela, tends to occur after pulmonary infection with the intracellular pathogen Mycobacterium tuberculosis (Mtb). Timely and accurate detection of A. fumigatus infection of pulmonary TB patients would undoubtedly greatly improve patient prognosis. Currently, the galactomannan (GM) antigen test is commonly used to detect A. fumigatus infection but has poor sensitivity that renders this assay inadequate for use in clinical practice.Design or Methods: Given the fact CPA and TB induce different host immune responses, we evaluated serum cytokine level profiles of CPA, TB patients and patients with both diseases (CPA-TB) for multiple cytokines and cytokine combinations.Results: The results revealed significantly higher serum levels of numerous proinflammatory cytokines, including IL-1β, IL-6, IL-8, IL-12p70, IFN-α, IFN-γ and TNF-α, in peripheral blood of CPA-TB patients versus that of TB patients. IL-8 levels alone provided the best discriminatory performance for distinguishing between TB and either CPA-TB patients (AUC = 0.949) or CPA patients (AUC = 0.964). Moreover, both IL-8 and TNF-α (AUC = 0.996) levels could be used to distinguish between TB and CPA-TB patients. Likewise, IL-8, TNF-α and IL-6 levels together could be used to distinguish between CPA-TB and TB patients.Conclusion: In this study, multiple cytokines were identified that may serve as potential biomarkers for use in detecting TB patients with CPA. Furthermore, our results should enhance understanding of how immune system dysfunctions influence susceptibility to Mtb and/or A. fumigatus infections.Keywords: Aspergillus fumigatus, Mycobacterium tuberculosis, cytokine profiles, IL-8, galactomannan

Chen Liang,1,2,* Shanshan Li,1,* Jinfeng Yuan,1 Yanhua Song,3 Weicong Ren,1 Wei Wang,1 Yuanyuan Shang,1 Shenjie Tang,2 Yu Pang1 1Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University /Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 2Tuberculosis Clinical Medical Center, Beijing Chest Hospital, Capital Medical University /Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 3Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shenjie Tang; Yu Pang, Beijing Chest Hospital, Capital Medical University, No. 97, Machang, Tongzhou District, Beijing, 101149, People’s Republic of China, Tel/Fax +86 010 8950 9367 ; +86 010 8950 9359, Email tangsj1106@vip.sina.com; pangyupound@163.comPurpose: This study aimed to investigate the phenotype, proliferation and functional alterations of cytokine-induced memory-like natural killer (CIML NK) cells from healthy subjects and TB patients, and assessed the efficacy of CIML NK cells in response to H37Rv-infected U937 cells in vitro.Methods: Fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy people and tuberculosis patients and activated for 16h using low-dose IL-15, or IL-12, IL-15, IL-18 combination or IL-12, IL-15, IL-18 and MTB H37Rv lysates, respectively, followed by low-dose IL-15 maintenance for another 7 days. Then, the PBMCs were co-cultured with K562 and H37Rv-infected U937, and the purified NK cells were co-cultured with H37Rv infected U937. The phenotype, proliferation and response function of CIML NK cells were assessed using flow cytometry. Finally, colony forming units were enumerated to confirm the survival of intracellular MTB.Results: CIML NK phenotypes from TB patients were similar to healthy controls. CIML NK cells undergo higher rates of proliferation after IL-12/15/18 pre-activation. Moreover, the poor expansion potential of CIML NK cells co-stimulated with MTB lysates. CIML NK cells from healthy individuals showed enhanced IFN-γ functional to H37Rv infected U937 cells, along with significantly enhanced killing of H37Rv. However, the CIML NK cells from TB patients show attenuated IFN-γ production and now enhanced the ability of killing intracellular MTB compared to those from healthy donors after co-cultured with H37Rv infected U937.Conclusion: CIML NK cells from healthy individuals exist the increased ability of IFN-γ secretion and boosted anti-MTB activity in vitro, which from TB patients show impaired IFN-γ production and no enhanced anti-MTB activity compared to those from healthy donors. Additionally, we observe the poor expansion potential of CIML NK cells co-stimulated with antigens from MTB. These results open up new possibilities for NK cell-based anti-tuberculosis immunotherapeutic strategies.Keywords: Mycobacterium tuberculosis, cytokines, NK cells, memory-like NK cells, IFN-&#x03B3

Tianhui Gao,1,&ast; Cong Yao,1,&ast; Yuanyuan Shang,1 Renchun Su,1 Xuxia Zhang,1 Weicong Ren,1 Shanshan Li,1 Wei Shu,2 Yu Pang,1 Qi Li2 1Department of Bacteriology and Immunology, Beijing Key Laboratory for Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People’s Republic of China; 2Clinical Center on Tuberculosis Control, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qi Li; Yu Pang, Beijing Chest Hospital, Capital Medical University, No. 97, Machang, Tongzhou District, Beijing, 101149, People’s Republic of China, Tel/Fax +86 010 6954 6690 ; +86 10 8950 9366, Email lq0703@hotmail.com; pangyupound@163.comPurpose: Unsatisfactory efficacies of currently recommended anti-Mycobacterium abscessus complex (MABC) treatment regimens have led to development of novel drugs to combat MABC infections. In this study, we evaluated in vitro antimicrobial activities of bedaquiline (BDQ) and four oxazolidinones against MABC isolates.Methods: The resazurin microplate assay was performed to determine minimum inhibitory concentrations (MICs) of BDQ and four oxazolidinones, including tedizolid (TZD), sutezolid (SZD), delpazolid (DZD), and linezolid (LZD), against 65 MABC isolates. A checkerboard method was used to investigate efficacies of various antimicrobial drug combinations.Results: BDQ MICs for MABC isolates ranged from < 0.031 to 1 μg/mL, while MIC50 and MIC90 values were 0.125 μg/mL and 0.25 μg/mL, respectively. TZD MIC50 and MIC90 values for MABC isolates were 1 μg/mL and 4 μg/mL, respectively, which were fourfold lower than corresponding LZD values (P < 0.001). DZD MIC90 values for MABC isolates was 8 μg/mL, which were 0.5-fold lower than corresponding LZD values (P < 0.01). MICs of BDQ, SZD, and LZD for M. abscessus subspecies massiliense isolates were significantly lower than corresponding MICs for M. abscessus subspecies abscessus isolates (P < 0.05). Notably, use of oxazolidinones (DZD, SZD, LZD, or TZD) with BDQ against MABC isolates led to reduction of the oxazolidinone median MIC range from 4 to 0.125 μg/mL to 1– 0.031 μg/mL.Conclusion: These results demonstrated excellent BDQ inhibitory activity against MABC isolates. TZD exhibited stronger antimicrobial efficacy against MABC isolates as compared to efficacies of DZD, SZD, and LZD. Importantly, MICs of oxazolidinones were markedly decreased when they were combined with BDQ, thus suggesting that combinations of BDQ and oxazolidinones may be effective treatments for MABC infections.Keywords: bedaquiline, tedizolid, oxazolidinones, susceptibility testing, Mycobacterium abscessus complex, delpazolid

Tuberculous meningitis (TBM), the most lethal and disabling form of tuberculosis (TB), may be related to gut microbiota composition, warranting further study. Here we systematically compared gut microbiota compositions and blood cytokine profiles of TBM patients, pulmonary TB patients, and healthy controls. Notably, the significant gut microbiota dysbiosis observed in TBM patients was associated with markedly high proportions of Escherichia-Shigella species as well as increased blood levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Next, we obtained a fecal bacterial isolate from a TBM patient and administered it via oral gavage to mice in order to develop a murine gut microbiota dysbiosis model for use in exploring mechanisms underlying the observed relationship between gut microbial dysbiosis and TBM. Thereafter, cells of commensal Escherichia coli (

Lin Qin,1,&ast; Fengmin Huo,2,&ast; Weicong Ren,2,&ast; Yuanyuan Shang,2 Cong Yao,2 Xuxia Zhang,2 Rongmei Liu,3 Liping Ma,3 Mengqiu Gao,3 Yu Pang2 1Department of Endoscopic Diagnosis & Treatment, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 2Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 3Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Mengqiu GaoDepartment of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel/Fax +86-10-8950 9322Email gaomqwdm@aliyun.comYu PangDepartment of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel/Fax +86-10-8950 9359Email pangyupound@163.comIntroduction: We assessed the effect of Mycobacterium tuberculosis (MTB) bacterial load on Xpert MTB/RIF accuracy for detection of rifampicin (RIF)-resistant MTB in bronchoalveolar lavage fluid (BALF) specimens obtained at a national tuberculosis (TB) specialized hospital in Beijing, China.Methods: A retrospective study was conducted at Beijing Chest Hospital. Patients with symptoms suggestive of pulmonary TB who provided BALF specimens for routine MTB detection between June 2019 and July 2020 were enrolled in the study. Chi-square test and Student’s t-test were used to compare results across groups stratified according to BALF bacterial load.Results: In total, 1125 patients with positive Xpert results who were enrolled in final analysis, 263 provided BALF specimens that tested positive for RIF-resistant MTB via Xpert MTB/RIF. The RIF-resistance rate of specimens with very low MTB bacterial load was 30.9%, a resistance rate significantly greater than rates obtained for groups with high (25.0%), medium (17.3%) and low (19.2%) MTB loads (P< 0.01). Notably, false-positive results obtained for the very low bacterial load group led to markedly reduced positive predictive value of Xpert MTB/RIF to provide correct RIF-resistance predictions for that group (67.1%, 95% CI: 56.1%– 78.1%5) relative to the predictive value obtained for all other groups combined (about 90%, P< 0.05). Sanger sequencing data obtained for 20 (32.8%) MTB isolates deemed RIF-resistant via Xpert (Probe E) lacked rpoB RRDR mutations. Meanwhile, of another group of 23 isolates deemed RIF-susceptible via DST but RIF-resistant via Xpert MTB/RIF, 20 isolate sequences (87.0%) lacked rpoB RRDR mutations, while sequences of the remaining 3 isolates harbored single rpoB RRDR mutations predicted to cause amino acid substitutions.Conclusion: Xpert MTB/RIF assay performed alarmingly poorly when used to detect RIF-resistant MTB in BALF specimens with very low bacterial loads. A high rate of Xpert probe E hybridization failure was the main driver of false-positive RIF-resistant results.Keywords: rifampicin, bronchoalveolar lavage fluid, Xpert MTB/RIF, China

Background & Objectives Accurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings. Methods A total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant. Results Overall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity. Conclusions We propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

The emergence of Mycobacterium abscessus complex (MABC) infection is the most noteworthy health care problem. Clarithromycin (CLA) and amikacin (AMK) constitute the cornerstone of treatment for patients infected with MABC; thus, early detection of resistance to these two drugs is essential for formulating effective therapeutic regimens. In the present study, we aimed to validate the use of MeltPro MAB assay, a melting curve analysis with dually labeled probes, on a set of clinical isolates to detect CLA and AMK resistance. A total of 103 clinical MABC strains were collected in our analysis, including 76 strains of M. abscessus

We aimed to determine the breakpoint of cycloserine (CS) susceptibility in MGIT and to describe the molecular characteristics of CS-resistant Mycobacterium tuberculosis (MTB) isolates.A total of 124 MTB isolates were recruited in our analysis. Minimum inhibitory concentration (MIC) was determined using the MGIT system. The mutations of MTB isolates within alr, ddl, ald, and cycA, potentially conferring CS resistance were analyzed by the whole-genome sequencing.In vitro drug susceptibility testing of isolates with doubling concentrations of CS revealed that the modal MIC values was 4 mg/L for MGIT, accounting for 35.5% (44/124) of isolates tested. Seven isolates harbored mutations conferring CS resistance, consisting of five with alr mutations and two with ald mutations. On the basis of the MIC distributions of wild-type and resistotype populations, we proposed a tentative epidemiologic cut-off value of 16 mg/l. The proportion of CS resistance in extensively drug-resistant TB was significantly higher than that of multidrug-resistant TB.In conclusion, we propose critical concentration for MGIT 960 to properly diagnose CS-resistant MTB and demonstrate that mutations in alr and ald genes are the major mechanism conferring CS resistance in clinical isolates.

Objective WFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. Methods We selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. Results MFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. Conclusion WFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.

The epidemic of pulmonary tuberculosis (TB), especially rifampin-resistant tuberculosis (RR-TB) presents a major challenge for TB control today. However, there is a lack of reliable and specific biomarkers for the early diagnosis of RR-TB. We utilized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to profile the transcript levels of 72 tripartite motif (TRIM) genes from a discovery cohort of 10 drug-sensitive tuberculosis (DS-TB) patients, 10 RR-TB patients, and 10 healthy controls (HCs). A total of 35 differentially expressed genes (DEGs) were screened out, all of which were down-regulated. The bio functions and pathways of these DEGs were enriched in protein ubiquitination, regulation of the viral process, Interferon signaling, and innate immune response, etc. A protein-protein interaction network (PPI) was constructed and analyzed using STRING and Cytoscape. Twelve TRIM genes were identified as hub genes, and seven (TRIM1, 9, 21, 32, 33, 56, 66) of them were verified by RT-qPCR in a validation cohort of 95 subjects. Moreover, we established the RR-TB decision tree models based on the 7 biomarkers. The receiver operating characteristic (ROC) analyses showed that the models exhibited the areas under the curve (AUC) values of 0.878 and 0.868 in discriminating RR-TB from HCs and DS-TB, respectively. Our study proposes potential biomarkers for RR-TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of RR-TB.