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Abstract Mycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection.

The most frequent adverse event associated with bedaquiline (BDQ) is the QTc interval prolongation; however, there was no biomarkers that could be used to predict the occurrence of QTc prolongation in BDQ-treated patients. In this study, we employed the ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) to generate metabolic profiling for the discovery of potential predictive urine biomarkers of QTc prolongation in these patients. Untargeted metabolomic technique was used to concentrate the differential metabolic pathway, and targeted metabolomic technique was subsequently performed to identify predictive biomarkers for QTc prolongation. A total of 45 rifampicin-resistant TB (RR-TB) and multidrug-resistant TB (MDR-TB) patients were enrolled in our study, including 15 RR/MDR-TB patients with QTc interval prolongation (QIP) and 30 RR/MDR-TB patients with QTc interval un-prolongations (QIU). Untargeted technique revealed that the lipid metabolism was the most differential metabolic pathway between two groups. Further targeted technique identified four differential metabolites, including betaine, LPE (18:2), LPE (20:3), and LPE (20:4). The combined analysis of metabolisms revealed that the combined use of LPE (20:3) and LPE (20:4) had the best performance for predicting the occurrence of QTc prolongation in TB patients, yielding a sensitivity of 87.4% and a specificity of 78.5%. In addition, with the progression of BDQ treatment, the LPEs exhibited persistent difference in the BDQ-treated TB patients experiencing QTc interval prolongation. In conclusion, our data demonstrate that the combined use of LPE (20:3) and LPE (20:4) yields promising performance for predicting the occurrence of QTc interval prolongation in BDQ-treated patients.

Abstract 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.

BackgroundLatent 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.MethodsParticipants 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.ResultsA 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.ConclusionThe 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.

Abstract Objectives Antimicrobial susceptibility tests (ASTs) are pivotal tools for detecting and combating infections caused by multidrug-resistant rapidly growing mycobacteria (RGM) but are time-consuming and labor-intensive. Design We used a Mycobacterium abscessus-based RGM model to develop a rapid (24-h) AST from the beginning of the strain culture, the Clinical Antimicrobials Susceptibility Test Ramanometry for RGM (CAST-R-RGM). The ASTs obtained for 21 clarithromycin (CLA)-treated and 18 linezolid (LZD)-treated RGM isolates. Results CAST-R-RGM employs D2O-probed Raman microspectroscopy to monitor RGM metabolic activity, while also revealing bacterial antimicrobial drug resistance mechanisms. The results of clarithromycin (CLA)-treated and linezolid (LZD)-treated RGM isolates exhibited 90% and 83% categorical agreement, respectively, with conventional AST results of the same isolates. Furthermore, comparisons of time- and concentration-dependent Raman results between CLA- and LZD-treated RGM strains revealed distinct metabolic profiles after 48-h and 72-h drug treatments, despite similar profiles obtained for both drugs after 24-h treatments. Conclusions Ultimately, the rapid, accurate, and low-cost CAST-R-RGM assay offers advantages over conventional culture-based ASTs that warrant its use as a tool for improving patient treatment outcomes and revealing bacterial drug resistance mechanisms.

Abstract Background Dendritic cells (DCs) are most potent antigen-processing cells and play key roles in host defense against Mycobacterium tuberculosis (MTB) infection. In this study, hub genes in DCs during MTB infection were first investigated using bioinformatics approaches and further validated in Monocyte-derived DCs. Methods Microarray datasets were obtained from Gene Expression Omnibus (GEO) database. Principal component analysis (PCA) and immune infiltration analysis were performed to select suitable samples for further analysis. Differential analysis and functional enrichment analysis were conducted on DC samples, comparing live MTB-infected and non-infected (NI) groups. The CytoHubba plugin in Cytoscape was used to identify hub genes from the differentially expressed genes (DEGs). The expression of the hub genes was validated using two datasets and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in human monocyte-derived DCs. Enzyme-linked immunosorbent assay (ELISA) was used to validate interferon (IFN) secretion. Transcription factors (TFs) and microRNAs (miRNAs) that interact with the hub genes were predicted using prediction databases. The diagnostic value of the hub genes was evaluated using receiver operating characteristic (ROC) curves and area under the curve (AUC) values. Results A total of 1835 common DEGs among three comparison groups (18 h, 48 h, 72 h after MTB infection) were identified. Six DEGs (IFIT1, IFIT2, IFIT3, ISG15, MX1, and RSAD2) were determined as hub genes. Functions enrichment analysis revealed that all hub genes all related to IFN response. RT-qPCR showed that the expression levels of six hub genes were significantly increased after DC stimulated by live MTB. According to the results of ELISA, the secretion of IFN-γ, but not IFN-α/β, was upregulated in MTB-stimulated DCs. AUC values of six hub genes ranged from 84 to 94% and AUC values of 5 joint indicators of two hub genes were higher than the two hub genes alone. Conclusion The study identified 6 hub genes associated with IFN response pathway. These genes may serve as potential diagnostic biomarkers in tuberculosis (TB). The findings provide insights into the molecular mechanisms involved in the host immune response to MTB infection and highlight the diagnostic potential of these hub genes in TB.

Background: In this study, we utilized whole genome sequencing (WGS) of clinical extremely drug-resistant tuberculosis (EDR-TB) strains collected during 2014–2020 in Beijing to detect clustered strains. Methods: A retrospective cohort study was conducted by inclusion of EDR-TB patients with positive cultures in Beijing between 2014 and 2020. Results: A total of 95 EDR-TB patients were included in our analysis. Up on the WGS based genotyping, 94 (94/95, 98.9%) out of 95 were identified as lineage 2 (East Asia). The pairwise genomic distance analysis identified 7 clusters, ranging in size from 2 to 5 isolates. The clustering rate of EDR-TB was 21.1%; while no patients had significantly higher odds of clustering. All isolates harbor rpoB RRDR mutations that confer RIF resistance and katG or inhA promoter mutations that confer INH resistance. Of 95 EDR-TB isolates, a total of 15 mutation types were recorded in the transcriptional regulator mmpR5. In vitro susceptibility testing results revealed that 14 (14/15, 93.3%) out of 15 mutation types were resistant to CFZ; whereas only 3 (3/15, 20.0%) showed resistance to BDQ. Interestingly, 12 isolates harbored mutations within rrl locus, whereas only mutations at positions 2294 and 2296 conferred CLA resistance. Favorable outcomes of EDR-TB patients were positively associated with more effective drugs in the regimes. Conclusion: WGS data demonstrate limited transmission of EDR-TB in this metropolis city. WGS-based drug susceptibility predictions will bring benefits to EDR-TB patients to formulate optimal therapeutic regimens.

Abstract 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.

ABSTRACT: Objectives: 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. Methods: 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. Results: 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. Conclusion: 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.

ObjectiveTuberculosis 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.MethodsThe 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.ResultsThe 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%).ConclusionThe 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.

Gazing is important during communication, which is a type of body language that differs from culture to culture. The present study investigated the influence of direct and averted gaze directions on the perceived duration of gazing among same-race and other-race faces. The gaze direction effect, in which direct-gazing faces were perceived to be longer than averted-gazing faces were, was found in the same-race condition but not in the other-race condition. The results could promote our understanding of the underlying mechanism of the gaze direction effect based on the perception of interpersonal social interaction.

ABSTRACT 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.

BackgroundApart from bactericidal effects, anti-tuberculosis drugs can interfere with the host’s immune system. In this study, we analyzed the role of delamanid (DLM), an inhibitor of mycolic acid synthesis of mycobacterial cell wall, on human macrophages.MethodsBased on a cohort of multidrug-resistant tuberculosis (MDR-TB) patients treated with DLM, the levels of C-reaction protein (CRP) and cytokines in the plasma were monitored using immunoturbidimetric assay and flow cytometry, respectively. We investigated the role of DLM on CXCL10 expression in U937 cell model using the following methods: cell viability assay, reverse transcription-quantitative polymerase chain reaction, enzyme linked immunosorbent assay, immunoblot, and transwell co-culture assay.ResultsA total of 23 MDR-TB patients were included, comprising of 13 patients treated with optimized background therapeutic regimen (OBR) plus DLM regimen (OBR+DLM) and 10 patients treated with OBR plus placebo. DLM administration was associated with a significant reduce in circulating CRP level. Correspondingly, after treatment, the level of CXCL10 in patients treated with OBR+DLM was significantly lower than that with control. Using cell model, DLM dramatically suppressed CXCL10 expression, which majorly depended on inhibiting the JAK/STAT pathway, and impaired the migration of PBMCs.ConclusionOur data firstly demonstrate that DLM suppresses CXCL10 expression via regulation of JAK2/STAT1 signaling and correlates with reduced inflammation in MDR-TB patients. DLM could be used as a potential drug for immunotherapy of patients with overactive immune response due to CXCL10.

Natural products from microorganisms are important sources for drug discovery. With the development of high-throughput sequencing technology and bioinformatics, a large amount of uncharacterized biosynthetic gene clusters (BGCs) in microorganisms have been found, which show the potential for novel natural product production. Nine BGCs containing PKS and/or NRPS in Streptomyces globisporus C-1027 were transcriptionally low/silent under the experimental fermentation conditions, and the products of these clusters are unknown. Thus, we tried to activate these BGCs to explore cryptic products of this strain. We constructed the cluster-situated regulator overexpressing strains which contained regulator gene(s) under the control of the constitutive promoter ermE*p in S. globisporus C-1027. Overexpression of regulators in cluster 26 resulted in significant transcriptional upregulation of biosynthetic genes. With the separation and identification of products from the overexpressing strain OELuxR1R2, three ortho-methyl phenyl alkenoic acids (compounds 1–3) were obtained. Gene disruption showed that compounds 1 and 2 were completely abolished in the mutant GlaEKO, but were hardly affected by deletion of the genes orf3 or echA in cluster 26. The type II PKS biosynthetic pathway of chain-extended cinnamoyl compounds was deduced by bioinformatics analysis. This study showed that overexpression of the two adjacent cluster-situated LuxR regulator(s) is an effective strategy to connect the orphan BGC to its products.

False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1β, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals [271 TB patients and 36 individuals with latent TB infection (LTBI)], demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting Mycobacterium tuberculosis (MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.

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.

ABSTRACT 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 subsp. Abscessus (MAA) and 27 strains of M. abscessus subsp. Massiliense (MAM). In vitro susceptibility testing revealed that two isolates exhibited intrinsic CLA resistance by harboring A2270T mutation in rrl, and inducible resistance was noted in 42 isolates. Additionally, two MAA isolates with erm(41)T28 genotype were susceptible to CLA. Notably, we found three out of 44 isolates had two melting curve peaks, representing the simultaneous presence of mutant and the wild type in these specimens. In contrast, no known mutations were identified in six AMK-resistant isolates. Further analysis revealed that MeltPro yielded 100% and 96.67% sensitivity and specificity for detecting CLA resistance. In summary, this study firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates, which significantly improves the turnaround time within 2 h. Approximate two fifths of MABC isolates are resistant to CLA by 23S rRNA mutation or its methylation, emphasizing the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections. IMPORTANCE Mycobacterium abscessus complex (MABC) has attracted increasing attention due to the numerous cases of infection. This pathogen is notorious for its intrinsic drug resistance, which complicates clinical management of patients with MABC infections. Clarithromycin (CLA) and amikacin (AMK) are the cornerstone of treatment regimens for MABC. Herein, our data firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates. The high frequency of CLA-resistant MABC isolates in China emphasizes the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections.

A reduction in visual performance is among the serious consequences of acute alcohol intake. Contrast sensitivity (CS) is a fundamental feature of visual function. Here, we investigated the negative effect of a moderate dose of alcohol on CS across a wide range of spatial frequencies and with multiple levels of external noise and clarified the corresponding mechanisms in the context of a perceptual template model (PTM). To avoid the effect of alcohol washout, a quick contrast sensitivity function (qCSF) method was used to assess the visual performance of subjects before and 30 min after a moderate dose of alcohol intake. We found that (1) CS was significantly disrupted by acute alcohol intake; (2) alcohol-induced CS loss was dependent on spatial frequency and external noise; and (3) increased internal additive noise and an impaired perceptual template accounted for alcohol-induced CS loss. These results help us better understand the negative effect of alcohol consumption and provide guidance for driver safety studies.

It is critical to address the relationship between density label of Bangerter filters and expected visual acuity, and how filters modulate contrast sensitivity (CS) at different spatial frequency and external noise levels. In the current study, the monocular visual acuity and CS at ten spatial frequencies and three noise levels were measured in normal subjects wearing no filters, 0.8, 0.4, or 0.2 Bangerter filters. Compared with the baseline condition (no filter worn), Bangerter filters degraded both visual acuity and contrast sensitivity function (CSF) in zero-noise conditions, but the reduction of both visual functions did not correlate with each other at any filter level. In addition, the stronger the filter was, the worse both visual functions became. In contrast, when external noise was present, filters improved the contrast sensitivity at low frequencies but deteriorated it at intermediate and high spatial frequencies. The perceptual template model was used to reveal the corresponding mechanism accounted for filter-induced visual function changes. Although the internal process in visual system should not be affected by the filters, the measurement of parameters was biased. To be specific, (1) the internal additive noise was elevated at all frequencies; (2) the perceptual template was improved at low spatial frequencies but impaired at intermediate spatial frequencies; and (3) the changes in both factors were highly dependent on filter intensity. We conclude that Bangerter filters influence visual acuity and contrast sensitivity differently and that their effect on contrast sensitivity depends on spatial frequency and noise.

ObjectivesIn this study, we conducted a systematic review to determine tuberculosis (TB) incidence due to immunotherapy with programmed cell death protein-1 (PD-1)/PD ligand (PD-L1) blockade in cancer patients.MethodsWe searched PubMed, Cochrance Library, Excerpt Medica Database (Embase), ClinicalTrials.gov, Chinese BioMedical Literature Database (CBM), China National Knowledge Infrastructure Database (CNKI), Wanfang and China Science and Technology Journal Database to identify studies between January 1, 2000 and April 30, 2021, on the reports of TB cases in patients treated with PD-1/PD-L1 blockade. Methodological quality of eligible studies was assessed, and random-effect model meta-analysis was performed to generate the pooled incidence estimate of TB cases in patients undergoing PD-1/PD-L1 therapy.ResultsWe initially identified 745 records, of which 27 studies ultimately met the inclusion criteria and were included in our meta-analysis. A total of 35 TB cases occurred among patients treated with PD-1/PD-L1 blockade. Nivolumab (51.4%) was the most frequently used PD-1/PD-L1 blockade for cancer treatment. In addition, pulmonary TB was the most common form of tuberculosis seen in 77.1% cases. Clinical outcomes were recorded in 18 patients, of whom 77.8% were cured or achieved remission, and 22.2% were died of TB. Pooled analysis determined that the TB rate in this population was 2,000 cases per 100,000 persons, and the estimated rate for TB associated with PD-1/PD-L1 blockade was 35 times higher than that in the general population.ConclusionTo conclude, our results demonstrate that the clinical use of PD-1/PD-L1 inhibitors significantly increases risk of TB reactivation. An extremely high mortality rate due to TB disease is noted in the patients with PD-1/PD-L1 blockade.

In this study, our aims were to comparatively analyze the power of variable number tandem repeat (VNTR) typing to discriminate isolates within subspecies and to identify a potential genetic marker for better molecular typing of Mycobacterium abscessus complex (MABC) strains. A total of 103 clinical MABC isolates were collected from a nationwide cross-sectional study in China. Eighteen VNTR loci were chosen to genotype the MABC isolates. Of the 103 clinical MABC isolates, there were 76 (73.8%) M. abscessus subsp. abscessus (MAA) and 27 (26.2%) M. abscessus subsp. massiliense (MAM) isolates. Among the patients with MAA lung diseases, the percentage of patients older than 45 years (67.1%) was significantly higher than that of patients with MAM lung diseases [33.3%, adjusted odds ratio (aOR) = 0.36, 95% CI = 0.13–0.98, p = 0.046]. Fifteen VNTR loci were designated as being “highly discriminant” in our sample, except for TR109. The total of 103 MABC isolates were fully discriminated into 103 unique patterns by an 18-locus VNTR set [Hunter–Gaston Discriminatory Index (HGDI) = 1.000], of which the inclusion of the top 12 loci yielded a comparative HGDI value (HGDI = 0.9998). Remarkably, the order of the diversity of the VNTR loci showed significant difference between the MAA and MAM isolates. TR137 and TR2, two loci with high diversity indices for the MAA isolates, only yielded poor discriminatory power for the MAM isolates; the allelic diversity (h) values were 0.0000 and 0.2621, respectively. A detailed analysis of TR137 in combination with the other 17 VNTR loci showed that the combination of TR137–TR2 could fully distinguish MAA from MAM isolates. In conclusion, our data revealed that MAA is more prone to affect elderly patients. Additionally, the population structure of the MABC isolates circulating in China has high diversity. The combined use of the TR137 and TR2 loci provides a simple criterion for the precise identification of MABC to the subspecies level.

Binocular summation is generally defined as the superiority of binocular over monocular performance. Here, we investigated how external noise modulates the effect of binocular summation on the contrast sensitivity function (CSF) and clarified the corresponding mechanisms with a perceptual template model (PTM). The contrast sensitivity (CS) over 10 spatial frequencies and three external noise levels was assessed under one binocular and two monocular viewing conditions. The binocular summation ratio (BSR) was calculated by dividing the area under the log CSF (AULCSF), or the CS of using both eyes, by that of only using the “good eye” (BSRG) or the “bad eye” (BSRB), respectively. We found that: (1) based on the AULCSF, the BSRB was higher than the BSRG; (2) based on the AULCSF, the BSR was more pronounced under zero-noise than under low-noise conditions, but the BSR was not higher than 1 under high-noise conditions due to a large individual difference; (3) based on the CS, with increasing spatial frequencies, the BSRB steadily increased; (4) both decreased internal additive noise and an improved perceptual template accounted for the gain in binocular summation. These results help us better understand the features of binocular CS and shed light on the clinical studies on populations with monocular CS loss.

Abstract Background Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC–MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. Results The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. Conclusions A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the “cryptic” herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

Source memory is one of the cognitive abilities that are most vulnerable to aging. Luckily, the brain plasticity could be modulated to counteract the decline. The repetitive transcranial magnetic stimulation (rTMS), a relatively non-invasive neuro-modulatory technique, could directly modulate neural excitability in the targeted cortical areas. Here, we are interested in whether the application of rTMS could enhance the source memory performance in healthy older adults. In addition, event-related potentials (ERPs) were employed to explore the specific retrieval process that rTMS could affect. Subjects were randomly assigned to either the rTMS group or the sham group. The rTMS group received 10 sessions (20 min per session) of 10 Hz rTMS applying on the right dorsolateral prefrontal cortex (i.e., F4 site), and the sham group received 10 sessions of sham stimulation. Both groups performed source memory tests before and after the intervention while the electroencephalogram (EEG) was recorded during the retrieval process. Behavioral results showed that the source memory performance was significantly improved after rTMS compared with the sham stimulation; ERPs results showed that during the retrieval phase, the left parietal old/new effect, which reflected the process of recollection common to both young and old adults, increased in the rTMS group compared with the sham stimulation group, whereas the late reversed old/new effect specific to the source retrieval of older adults showed similar attenuation after intervention in both groups. The present results suggested that rTMS could be an effective intervention to improve source memory performance in healthy older adults and that it selectively facilitated the youth-like recollection process during retrieval. This study was registered in the Chinese Clinical Trial Registry (ChiCTR) with the identifier chictr-ire-15006371.

It is well known that extensive practice of a perceptual task can improve visual performance, termed perceptual learning. The goal of the present study was to evaluate the dependency of visual improvements on the features of training stimuli (i.e., spatial frequency). Twenty-eight observers were divided into training and control groups. Visual acuity (VA) and contrast sensitivity function (CSF) were measured and compared before and after training. All observers in the training group were trained in a monocular grating detection task near their individual cutoff spatial frequencies. The results showed that perceptual learning induced significant visual improvement, which was dependent on the cutoff spatial frequency, with a greater improvement magnitude and transfer of perceptual learning observed for those trained with higher spatial frequencies. However, VA significantly improved following training but was not related to the cutoff spatial frequency. The results may broaden the understanding of the nature of the learning rule and the neural plasticity of different cortical areas.

Increasing evidence suggests that functional brain connectivity is an important determinant of cognitive aging. However, the fundamental concept of inter-individual variations in functional connectivity in older individuals is not yet completely understood. It is essential to evaluate the extent to which inter-individual variability in connectivity impacts cognitive performance at an older age. In the current study, we aimed to characterize individual variability of functional connectivity in the elderly and to examine its significance to individual cognition. We mapped inter-individual variability of functional connectivity by analyzing whole-brain functional connectivity magnetic resonance imaging data obtained from a large sample of cognitively normal older adults. Our results demonstrated a gradual increase in variability in primary regions of the visual, sensorimotor, and auditory networks to specific subcortical structures, particularly the hippocampal formation, and the prefrontal and parietal cortices, which largely constitute the default mode and fronto-parietal networks, to the cerebellum. Further, the inter-individual variability of the functional connectivity correlated significantly with the degree of cognitive relevance. Regions with greater connectivity variability demonstrated more connections that correlated with cognitive performance. These results also underscored the crucial function of the long-range and inter-network connections in individual cognition. Thus, individual connectivity–cognition variability mapping findings may provide important information for future research on cognitive aging and neurocognitive diseases.

Hyperlipidemia, one of the cardiovascular (CV) risk factors, is associated with an increase in the risk for dementia. Repetitive transcranial magnetic stimulation (rTMS) was applied over the right dorsolateral prefrontal cortex (DLPFC) to modulate serum lipid levels in older adults. Participants received 10 sessions of rTMS or sham stimulation intervention within 2 weeks. The serum lipid and thyroid hormone-related endocrine levels were assessed before and after the treatment. We found that rTMS significantly decreased serum lipid levels, including the total cholesterol (CHO) and triglyceride (TG); meanwhile, it also increased the thyroid-stimulating hormone (TSH) as well as thyroxine (T4) levels. This suggests that rTMS modulated the serum lipid metabolism by altering activity in the hypothalamo-pituitary-thyroid (HPT) axis. The trial was registered on the website of Chinese Clinical Trial Registry (http://www.chictr.org.cn).