Antigen-specific chemokine profiles as biomarkers for detecting Mycobacterium tuberculosis infection.

Background: Latent tuberculosis (TB) infection can progress to active TB, which perpetuates community transmission that undermines global TB control efforts. Clinically, interferon-γ release assays (IGRAs) are commonly used for active TB case detection. However, low IGRA sensitivity rates lead to false-negative results for a high proportion of active TB cases, thus highlighting IGRA ineffectiveness in differentiating MTB-infected individuals from healthy individuals. Methods: Participants enrolled at Beijing Chest Hospital from May 2020-April 2022 were assigned to healthy control (HC), LTBI, IGRA-positive TB, and IGRA-negative TB groups. Screening cohort MTB antigen-specific blood plasma chemokine concentrations were measured using Luminex xMAP assays then were verified via testing of validation cohort samples. Results: A total of 302 individuals meeting study inclusion criteria were assigned to screening and validation cohorts. Testing revealed significant differences in blood plasma levels of CXCL9, CXCL10, CXCL16, CXCL21, CCL1, CCL19, CCL27, TNF-α, and IL-4 between IGRA-negative TB and HC groups. Levels of CXCL9, CXCL10, IL-2, and CCL8 biomarkers were predictive for active TB, as reflected by AUC values of ≥0.9. CXCL9-based enzyme-linked immunosorbent assay sensitivity and specificity rates were 95.9% (95%CI: 91.7-98.3) and 100.0% (92.7-100.0), respectively. Statistically similar AUC values were obtained for CXCL9 and CXCL9-CXCL10 assays, thus demonstrating that combined analysis of CXCL10 and CXCL9 levels did not improve active TB diagnostic performance. Conclusion: The MTB antigen stimulation-based CXCL9 assay may compensate for low IGRA diagnostic accuracy when used to diagnose IGRA-negative active TB cases and thus is an accurate and sensitive alternative to IGRAs for detecting MTB infection.

Prolonged Dual Antiplatelet Therapy in Acute Myocardial Infarction Patients Without Revascularization: China Acute Myocardial Infarction Registry Study.

At least 12 months of dual antiplatelet therapy (DAPT) is 1 of the standards of care following percutaneous coronary intervention in patients with acute coronary syndrome. However, study on prolonged DAPT for patients with acute myocardial infarction (AMI) without revascularization is limited. We studied 1,744 patients with AMI without revascularization from the China Acute Myocardial Infarction registry between January 2013 and September 2014. These patients were on DAPT and did not experience AMI, stroke, or bleeding events at the 12-month follow-up. We divided them into 2 groups: 12-month DAPT group (DAPT for at least 12 months but <18 months) and 18-month DAPT group (DAPT for at least 18 months). The primary outcome was 24-month all-cause death. Overall, 1,221 patients (70.0%) took DAPT for ≥12 months but <18 months, whereas 523 patients (30.0%) took DAPT for ≥18 months. The proportion of patients at high ischemic risk and the proportion of patients at high bleeding risk were similar in the 2 groups. At 24 months, the all-cause mortality rate of the 18-month DAPT group was significantly lower than that for the 12-month DAPT group (3.7% vs 5.9%, p = 0.0471). The adjusted hazard ratio for all-cause death also showed statistical significance (0.59, 95% confidence interval 0.35 to 0.99, p = 0.0444). In conclusion, DAPT for at least 18 months appears to be associated with lower 24-month mortality for non-revascularization AMI patients without events within 12 months after onset.

In vitro susceptibility of nontuberculous mycobacteria in China.

OBJECTIVES: This study aimed to measure the prevalence of resistance to antimicrobial agents, and explore the risk factors associated with drug resistance by using nontuberculous Mycobacteria (NTM) isolates from China. METHODS: A total of 335 NTM isolates were included in our analysis. Broth dilution method was used to determine in vitro drug susceptibility of NTM isolates. RESULTS: Clarithromycin (CLA) was the most potent drug for Mycobacterium intracellulare (MI). The resistance rate of 244 MI isolates to CLA was 21%, yielding a minimum inhibitory concentrations (MIC)50 and MIC90 of 8 and 64 mg/L, respectively. 51% of 244 MI isolates exhibited resistance to amikacin (AMK). For 91 Mycobacterium abscessus complex (MABC) isolates, 6 (7%) and 49 (54%) isolates were categorized as resistant to CLA at day 3 and 14, respectively. The resistance rate to CLA for Mycobacterium abscessus subspecies abscessus (MAA) was dramatically higher than that for Mycobacterium abscessus subspecies massiliense (MAM). Additionally, the percentage of patients presenting fever in the CLA-susceptible group was significantly higher than that in the CLA-resistant group. CONCLUSIONS: Our data demonstrate that approximate one fifth of MI isolates are resistant to CLA. We have identified a higher proportion of CLA-resistant MAA isolates than MAM. The patients caused by CLA-resistant MI are at low risk for presenting with fever relative to CLA-susceptible group.

The emerging threat of fluroquinolone-, bedaquiline-, and linezolid-resistant Mycobacterium tuberculosis in China: Observations on surveillance data.

BACKGROUND: Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB), constitutes a major obstacle to fulfill end TB strategy globally. Although fluoroquinolones (FQs), linezolid (LZD) and bedaquiline (BDQ) were classified as Group A drugs for MDR-TB treatment, our knowledge of the prevalence of TB which were resistant to Group A drugs in China is quite limited. METHODS: In this study, we conducted a prospective multicenter surveillance study in China to determine the proportion of TB patients that were resistant to Group A drugs. A total of 1877 TB patients were enrolled from 2022 at four TB specialized hospitals. The drug susceptibility of isolated strains was conducted using the MGIT 960 system and the molecular mechanisms conferring drug resistance were investigated by Sanger sequencing. RESULTS: 12.9% of isolates were resistant to levofloxacin (LFX), 13.2% were resistant to moxifloxacin (MOX), 0.2% were resistant to bedaquiline (BDQ), and 0.8% were resistant to linezolid (LZD). Totally, 14.0% and 0.4% were classified as multidrug resistant- (MDR-) and extensively drug resistant- (XDR-) TB. The drug resistance was more common in retreated TB cases compared to new cases. In addition, 70.0% of fluoroquinolone (FQ)-resistant isolates harbored mutations in the gyrA and gyrB gene. By contrast, the common drug-resistant mutations were only found in 50% BDQ-resistant and 20% LZD-resistant isolates. CONCLUSIONS: Our data demonstrate that approximate half of MDR -TB patients are resistant to fluoroquinolones, with extremely low prevalence of initial BDQ and LZD resistance. Findings from this study provide important implications for the current management of MDR-TB patients.

Induction of lysosomal exocytosis and biogenesis via TRPML1 activation for the treatment of uranium-induced nephrotoxicity.

Uranium (U) is a well-known nephrotoxicant which forms precipitates in the lysosomes of renal proximal tubular epithelial cells (PTECs) after U-exposure at a cytotoxic dose. However, the roles of lysosomes in U decorporation and detoxification remain to be elucidated. Mucolipin transient receptor potential channel 1 (TRPML1) is a major lysosomal Ca2+ channel regulating lysosomal exocytosis. We herein demonstrate that the delayed administration of the specific TRPML1 agonist ML-SA1 significantly decreases U accumulation in the kidney, mitigates renal proximal tubular injury, increases apical exocytosis of lysosomes and reduces lysosomal membrane permeabilization (LMP) in renal PTECs of male mice with single-dose U poisoning or multiple-dose U exposure. Mechanistic studies reveal that ML-SA1 stimulates intracellular U removal and reduces U-induced LMP and cell death through activating the positive TRPML1-TFEB feedback loop and consequent lysosomal exocytosis and biogenesis in U-loaded PTECs in vitro. Together, our studies demonstrate that TRPML1 activation is an attractive therapeutic strategy for the treatment of U-induced nephrotoxicity.

Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

Serum Cytokine Biomarkers for Use in Diagnosing Pulmonary Tuberculosis versus Chronic Pulmonary Aspergillosis.

Background: 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-1ß, 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.

Analysis of Xpert MTB/RIF results in retested patients with very low initial bacterial loads: A retrospective study in China.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay has been widely used to diagnose suspected active tuberculosis (TB) and rifampicin-resistant TB cases. Despite its excellent performance record, false-positive Xpert rifampicin (RIF) resistance results are obtained for specimens with extremely low bacterial loads. OBJECTIVE: We aimed to study the feasibility of repeat Xpert testing as a strategy for reducing the odds of obtaining false-positive results when testing paucibacillary TB patients. METHODS: We enrolled previously tested TB patients with very low initial bacterial loads from May 2016 to February 2022 for Xpert retesting. A total of 251 TB patients were retested using the Xpert assay. RESULTS: RIF resistance was noted in 65 (25.9 %) patients when tested by Xpert at initial diagnosis. Only 107 (42.6 %) of 251 patients tested positive for MTB when retested via Xpert. The majority (98.6 %) of RIF-susceptible cases were still susceptible to RIF when retested. Initial Xpert testing yielded 35 positive results for MTB in the RIF-resistant group, of whom 25 (71.4 %) still exhibited RIF resistance when retested. All culture-positive MTB isolates in the RIF-susceptible group were also RIF-susceptible by phenotypic DST. In the RIF-resistant group, 10 of 14 culture-positive MTB isolates exhibited RIF resistance, of which 4 isolates were deemed RIF-susceptible by phenotypic DST. The proportion of double mutations within the MTB rpoB RRDR sequence, as detected by hybridization of Xpert D and E probes, was significantly higher in the RIF-susceptible group than in the RIF-susceptible group. CONCLUSIONS: Our results demonstrated that initial RIF-susceptible results were more accurate than RIF-resistant results. Additionally, patients with double mutations that delayed probe D/E hybridization were more likely to have false-positive Xpert results. Our findings emphasize that repeat Xpert MTB/RIF testing is necessary for TB patients with extremely low bacterial loads who are at high risk for RIF-resistant TB.

Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens.

Objective: Tuberculosis diagnosis requires rapid, simple and highly sensitive methods. Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated protein (Cas) systems are increasingly being used for clinical diagnostic applications, due to their high flexibility, sensitivity and specificity. We developed a sensitive Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens. Methods: The conserved MTB IS1081 sequence was used to design CRISPR-derived RNAs (crRNAs) and T7 promoter sequencing-containing PCR primers for use in the CRISPR-MTB assay, then assay performance was evaluated using 401 clinical specimens. Results: The CRISPR-MTB assay provided a low limit of detection of 1 target sequence copy/µL and excellent specificity. Furthermore, use of the assay to detect MTB in bronchoalveolar lavage fluid (BALF), sputum and pus samples provided superior sensitivity (261/268, 97.4%) as compared to sensitivities of acid-fast bacilli (130/268, 48.5%) and mycobacterial culture (192/268, 71.6%) assays, and comparable or greater sensitivity to that of GeneXpert MTB/RIF (260/268, 97.0%). Conclusion: The CRISPR-MTB assay, which provides excellent sensitivity and specificity for MTB detection in sputum, BALF and pus samples, is a viable alternative to conventional tests used to diagnose TB in resource-limited settings.

Antimicrobial Effect of Oxazolidinones and Its Synergistic Effect with Bedaquiline Against Mycobacterium abscessus Complex.

Purpose: 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.

The Correlation Between 18F-Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography Semiquantitative Parameters and the Clinical Features and Pathological Biological Indexes of Gastric Cancer.

Objective: This study explored the application value of the maximum standard uptake value (SUVmax) of 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) in gastric cancer. Materials and Methods: Data of 164 patients with gastric cancer who had undergone18F-FDG PET/CT before a biopsy were collected, and the correlation of SUVmax with clinical stage, pathological differentiation degree, human epidermal growth factor receptor-2 (HER-2) status, and Ki-67 index of gastric cancer was analyzed. Results: The SUVmax of poorly differentiated adenocarcinoma was significantly higher than that of moderately differentiated adenocarcinoma and signet-ring cell carcinoma (p < 0.01), and SUVmax in the well-differentiated adenocarcinoma group was higher than that in the signet-ring cell carcinoma group (p < 0.01). The SUVmax in the HER-2 negative group was higher than that in the HER-2 positive group (p < 0.01). The SUVmax was higher in the Ki-67 high expression group than in the low expression group (p < 0.01), and there was a significant positive correlation between the two (p < 0.01). Conclusion: 18F-FDG PET/CT SUVmax can, to some extent, predict the degree of differentiation, HER-2 status, and Ki-67 index of gastric cancer patients.

Diagnostic accuracy of oral swab for detection of pulmonary tuberculosis: a systematic review and meta-analysis.

Objectives: Tuberculosis (TB) remains a significant concern in terms of public health, necessitating the timely and accurate diagnosis to impede its advancement. The utilization of oral swab analysis (OSA) presents a promising approach for diagnosing pulmonary TB by identifying Mycobacterium tuberculosis (MTB) within oral epithelial cells. Due to disparities in the diagnostic performance of OSA reported in the original studies, we conducted a meticulous meta-analysis to comprehensively assess the diagnostic efficacy of OSA in pulmonary TB. Methods: We conducted a comprehensive investigation across multiple databases, namely PubMed, Cochrane Library, Embase, Web of Science, ClinicalTrials.gov, Chinese BioMedical Literature Database (CBM), China National Knowledge Infrastructure Database (CNKI), and Wanfang China Science and Technology Journal Database to identify relevant studies. Out search query utilized the following keywords: oral swab, buccal swab, tongue swab, tuberculosis, and TB. Subsequently, we employed STATA 16.0 to compute the combined sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for both the overall and subgroup analyses. Results: Our findings indicated that OSA has a combined sensitivity of 0.67 and specificity of 0.95 in individuals with pulmonary TB. Subgroup analysis further revealed that among adult individuals with pulmonary TB, the sensitivity and specificity of OSA were 0.73 and 0.93, respectively. In HIV-negative individuals with pulmonary TB, the sensitivity and specificity were 0.68 and 0.98, respectively. The performance of OSA in detecting pulmonary TB correlated with the bacteria load in sputum. Additionally, the sensitivity for diagnosing pulmonary TB using tongue specimens was higher (0.75, 95% CI: 0.65-0.83) compared to cheek specimens (0.52, 95% CI: 0.34-0.70), while both types of specimens demonstrated high specificity. Conclusions: To conclude, oral swabs serve as a promising alternative for diagnosing pulmonary TB, especially in adult patients. In addition, tongue swabs yield better sensitivity than cheek swabs to identify pulmonary TB patients. Systematic review registration: identifier: CRD42023421357.

Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis.

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 (E. coli) were isolated and administered to model mice by gavage and then mice were inoculated with Mycobacterium tuberculosis (M. tuberculosis). Subsequently, these mice exhibited increased blood TNF-α levels accompanied by downregulated expression of tight junction protein claudin-5, increased brain tissue bacterial burden, and elevated central nervous system inflammation relative to corresponding indicators in controls administered PBS by gavage. Thus, our results demonstrated that a signature dysbiotic gut microbiome profile containing a high proportion of E. coli was potentially associated with an increased circulating TNF-α level in TBM patients. Collectively, these results suggest that modulation of dysbiotic gut microbiota holds promise as a new strategy for preventing or alleviating TBM. IMPORTANCE As the most severe form of tuberculosis, the pathogenesis of tuberculous meningitis (TBM) is still unclear. Gut microbiota dysbiosis plays an important role in a variety of central nervous system diseases. However, the relationship between gut microbiota and TBM has not been identified. In our study, significant dysbiosis in gut microbiota composition with a high proportion of E. coli and increased levels of TNF-α in plasma was noted in TBM patients. A commensal E. coli was isolated and shown to increase the plasma level of TNF-α and downregulate brain tight junction protein claudin-5 in the murine model. Gavage administration of E. coli aggravated the bacterial burden and increased the inflammatory responses in the central nervous system after M. tuberculosis infection. Dysbiosis of gut microbiota may be a promising therapeutic target and biomarker for TBM prevention or treatment.

A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation.

Various antibiotics such as tetracycline, aureomycin, amoxicillin, and levofloxacin are found in large quantities in groundwater and soil systems, potentially leading to the development of resistant and multi-drug resistant bacteria, posing a threat to humans, animals, and environmental systems. Photocatalytic technology has attracted keen interest due to its rapid and stable treatment and direct use of solar energy. However, most studies evaluating the performance of semiconductor catalysts for the photocatalytic degradation of organic pollutants in water are currently incomplete. In this paper, a complete experimental protocol is designed to comprehensively evaluate the photocatalytic performance of semiconductor catalysts. Herein, rhombic dodecahedral silver phosphate was prepared by a simple solvent phase synthesis method at room temperature and atmospheric pressure. BrSubphthalocyanine/Ag3PO4 heterojunction materials were prepared by the solvothermal method. The catalytic performance of as-prepared materials for the degradation of tetracycline was evaluated by studying different influencing factors such as catalyst dosage, temperature, pH, and anions at atmospheric pressure using a 300 W xenon lamp as a simulated solar light source and a light intensity of 350 mW/cm2. Compared with the first cycle, the constructed BrSubphthalocyanine/Ag3PO4 maintained 82.0% of the original photocatalytic activity after five photocatalytic cycles, while the pristine Ag3PO4 maintained only 28.6%. The stability of silver phosphate samples was further tested by a five-cycle experiment. This paper provides a complete process for evaluating the catalytic performance of semiconductor catalysts in the laboratory for the development of semiconductor catalysts with potential for practical applications.

Transition between Mycobacterium tuberculosis and nontuberculous mycobacteria in recurrent "tuberculosis" patients.

Recurrence of tuberculosis (TB) is still a key issue in the control of tuberculosis. The presence of nontuberculous mycobacteria (NTM) complicates the diagnosis of recurrent TB due to similarity in clinical presentation. Herein, we have used molecular genotyping methods to identify mycobacteria species, and analyzed the characteristics of patients with transition between MTB and NTM. Eighty-nine patients with recurrent tuberculosis over the past 12 years were included in our analysis. We found that 9 patients had NTM infections during the study period. Six patients were infected with different mycobacterial strains, half of which were transformed from NTM to MTB, and the other half from MTB to NTM. In addition, the other 3 patients were infected with the same NTM species. Further WGS analysis showed that only one patient had a relapse and the remaining two were classified as reinfection. In conclusion, our results demonstrate that a proportion of previously diagnosed recurrent TB cases are attributed to the transition between MTB and NTM, highlighting the significance of species identification prior to initiation of treatment. The recurrence of mycobacterial diseases is majorly noted within 1 year after treatment completion.

Reevaluating Rifampicin Breakpoint Concentrations for Mycobacterium tuberculosis Isolates with Disputed rpoB Mutations and Discordant Susceptibility Phenotypes.

In this study, rifampicin resistance breakpoints based on MICs of disrupted rpoB mutants of Mycobacterium tuberculosis (MTB) were explored using the Mycobacteria Growth Indicator Tube (MGIT) system and microplate alamarBlue assay (MABA). Sixty-one MTB isolates with disputed low-level rifampicin resistance-associated rpoB mutations and 40 RIF-susceptible wild-type isolates were included. Among the 61 resistant isolates, 25 (41.0%) had MICs ≥2.0 mg/L via MABA, while 16 (26.2%) were identified as RIF resistant via MGIT. Epidemiological cut-off (ECOFF) values obtained using MABA and MGIT were 0.25 and 0.125 mg/L, respectively. Based on 0.125 mg/L as a tentative critical concentration (CC), MABA RIF resistance-detection sensitivity was 93.4%, prompting the reduction of the MGIT CC to 0.125 mg/L, given that only a single isolate (1.6%) with the borderline mutation would be misclassified as susceptible to RIF based on this CC. Based on DNA sequencing of RRDR as the gold standard, the diagnostic accuracy of MGIT (99.0%) was significantly higher than that of MABA (91.1%). MICs of Leu511Pro mutant isolates were negatively correlated with time to liquid culture positivity (TTP) in our analysis (R = 0.957, P < 0.01). In conclusion, our results demonstrated missed detection of a high proportion of rifampicin-resistant isolates based on the WHO-endorsed CC. Such missed detections would be avoided by reducing the optimal MGIT RIF CC to 0.125 mg/L. In addition, MGIT based on reduced CC outperformed MABA in detecting borderline RIF resistance, with MABA MIC results obtained for isolates with the same mutation correlating with MTB growth rate. IMPORTANCE Tuberculosis (TB) is still one of the world's leading infectious disease killers. The early and accurate diagnosis of RIF resistance is necessary to deliver timely and appropriate treatment for TB patients and improve their clinical outcome. Actually, a proportion of MTB isolates with disputed rpoB mutations present a diagnostic dilemma between Xpert and phenotypical drug susceptibility testing (pDST). Recently, WHO reported a pragmatic approach by lowering critical concentration (CC) to boost sensitivity of resistance detection of pDST. Therefore, a detailed analysis of the association between RIF susceptibility and disrupted mutations within rpoB gene would lay a foundation to assess the diagnostic accuracy of pDST with lowering RIF CC. In this study, we aim to determine the MICs of MTB isolates with disrupted mutations by MGIT and microplate alamarBlue assay (MABA). We also aimed to determine the optimal breakpoints for MTB isolates with these mutations.

Effect of Mixed Infections with Mycobacterium tuberculosis and Nontuberculous Mycobacteria on Diagnosis of Multidrug-Resistant Tuberculosis: A Retrospective Multicentre Study in China.

BACKGROUND: Correct species identification is essential before initiation of TB treatment, due to substantial drug susceptibility profile differences among mycobacterial species. Given that nontuberculous mycobacteria (NTM) are frequently resistant to first-line anti-tuberculosis drugs, cases with mixed infections with Mycobacterium tuberculosis (MTB) and NTM tend to be diagnosed as multidrug-resistant tuberculosis (MDR-TB) cases. Here we report results of a retrospective multicentre study that was conducted to determine the prevalence of TB-NTM infections in previously diagnosed laboratory-confirmed multidrug-resistant tuberculosis (MDR-TB) patients using phenotypic drug susceptibility testing. The results were then used to identify risk factors associated with susceptibility to mixed infections. METHODS: From January 2019 through December 2019, we retrospectively collected MDR-TB isolates from three TB specialised hospitals. Species identifications of isolates were performed using the MeltPro Myco assay. RESULTS: A total of 837 MDR-TB isolates were analysed, of which 22 isolates (2.6%) were found to contain a mixture of NTM and MTB organisms. Significant differences in prevalence rates of mixed infections across regions were observed, with prevalence rates ranging from 0.0% (0/213) in Beijing to 3.4% (12/353) in Fuzhou to 3.7% (10/271) in Guangzhou. Among the 22 patients with NTM-TB mixed infections, a total of five different mycobacterial species were identified, of which the most prevalent species was Mycobacterium intracellulare. Notably, a history of previous TB episodes correlated with higher mixed infection risk. CONCLUSION: The results reported here demonstrated that mixed infections with MTB and NTM occurred in approximately 3% of suspected MDR-TB patients in China. These findings raise concerns about the accuracy of molecular diagnostics-based species identification tests and draw attention to the possibility that NTM-MTB mixed infections will be misdiagnosed as MDR-TB in high TB burden settings.

Clinical isolates of Mycobacterium tuberculosis with different genotypes exhibit distinct host macrophage responses in vitro.

Introduction. Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, can survive as an intracellular parasite after entering macrophages via phagocytosis. M.tb strains are genotypically distinct and engage in diverse pathogen-host interactions, with different host immune responses triggered by different M.tb strains. Importantly, differences in intracellular accumulation and triggering of host macrophage responses during early infection stages are key determinants that shape the final outcomes of host innate immune responses to different M.tb strains.Hypothesis/Gap Statement. Clinical M.tb strains with different genotypes elicit different host innate immune responses in vitro.Aim. This work aimed to compare host innate immune responses elicited by genotypically diverse, clinically derived M.tb strains in vitro.Methodology. RAW264.7 cells were infected with three lineage 2 and lineage 4 clinically derived M.tb strains and strain H37Rv. Strains were evaluated for differences in intracellular growth, induction of macrophage apoptosis, and induction of expression of proinflammatory cytokines and associated pattern recognition receptors.Results. Highly variable cytokine profiles were observed subsequent to RAW264.7 cell infection with the different strains. The Beijing genotype strain, a modern Beijing strain belonging to lineage 2, induced milder host proinflammatory responses and less apoptosis and exhibited greater intracellular growth as compared to the other strains. Moreover, mRNA expression levels of iNOS in Beijing and MANU2 genotype strains exceeded corresponding levels obtained for the T1 genotype strain. Meanwhile, mRNA expression levels of toll-like receptor (TLR)-encoding genes TLR2 and TLR7 in macrophages infected with the Beijing genotype strain were higher than corresponding levels observed in MANU2 genotype strain-infected macrophages.Conclusion. The higher intracellular survival rate and lower level of host cell apoptosis associated with macrophage infection with the Beijing genotype strain indicated greater virulence of this strain relative to that of the other strains. Furthermore, in vitro immune responses induced by the Beijing genotype strain were unique in that this strain induced a weaker inflammatory response than was induced by T1 or MANU2 genotype strains. Nevertheless, additional evidence is needed to confirm that Beijing genotype strains possess greater virulence than strains with other genotypes.

Distinguishing Relapse From Reinfection With Whole-Genome Sequencing in Recurrent Pulmonary Tuberculosis: A Retrospective Cohort Study in Beijing, China.

Background: Tuberculosis recurrence is still a major problem for the control of tuberculosis, and the cause of the recurrence is still unclear. Methods: We retrospectively recruited 68 pairs of samples of Mycobacterium tuberculosis (MTB) from recurrent TB cases in Beijing Chest Hospital between January 2008 and December 2019. The whole-genome sequencing was conducted to analyze single-nucleotide polymorphism (SNP) and to identify whether recurrent disease was due to relapse or reinfection. The BACTEC MGIT was performed to compare differences in drug susceptibility profiles between two episodes. Results: 62 (91.2%) out of 68 confirmed recurrence were due to relapse, whereas the remaining six (8.8%) were due to reinfection. And there was a strong association between earlier relapse and underlying chronic diseases. In addition, the MTB isolates from non-diabetic patients had a higher mutation rate than those from diabetic patients. A community transmission was also identified in our cohort. Levofloxacin resistance was the most frequently observed drug resistance for 12.9% relapse cases. Conclusion: The relapse of a previous episode in Beijing. The underlying chronic diseases are associated with an earlier TB relapse. MTB isolates were more prone to develop levofloxacin resistance than moxifloxacin resistance after FQ exposure. The patients at high-risk for relapses deserve more careful investigation.

Prevalence of extensively drug-resistant tuberculosis in a Chinese multidrug-resistant TB cohort after redefinition.

OBJECTIVES: Recently, the definition of extensively drug-resistant TB (XDR-TB) has been revised. In this study, we conducted a descriptive and retrospective study to determine the prevalence of XDR-TB in a Chinese multidrug-resistant TB (MDR-TB) cohort. METHODS: Broth microdilution method was performed to determine in vitro susceptibilities of Mycobacterium tuberculosis (MTB) isolates to (FQs), bedaquiline (BDQ) and linezolid (LZD). The putative drug target genes conferring drug resistance were screened by DNA sequencing. RESULTS: A total of 425 MDR-TB isolates were included from 13 pilots in China. LZD and BDQ resistance were noted in 30 (7.1%) and 10 (2.4%) isolates. On the basis of latest definitions, 114 (26.8%) were MDR-TB, 282 (66.4%) were pre-XDR-TB, and 29 (6.8%) were XDR-TB. Among 311 FQ-resistant isolates, 265 harbored genetic mutations within QRDRs. The most common mutations were observed at codon 94 of gyrA, accounting for 47.2% of FQ-resistant MTB isolates. Only mutations within the Rv0678 gene were found to confer BDQ resistance in our cohort, conferring 40.0% of BDQ resistance. For LZD resistance, 53.3% of LZD-resistant isolates carried genetic mutations in rplC or 23S rRNA. The most frequent mutation was Cys154Arg in the rplC gene. In addition, we recorded two MDR-TB patients with resistance to both BDQ and LZD, of which one patient experienced continuous positive culture of MTB despite inclusion of efficacious moxifloxacin. CONCLUSION: Our results demonstrate that the low prevalence of XDR-TB holds great promise for MDR-TB treatment with WHO-endorsed regimens containing BDQ-LZD combination, whereas the high prevalence of FQ-resistance in MDR-TB patients warrants national attention.