Single-cell transcriptomics of blood identified IFIT1+ neutrophil subcluster expansion in NTM-PD patients.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment. METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq. RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls. CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

Factors Influencing False-Negative Results of QuantiFERON-TB Gold In-Tube (QFT-GIT) in Active Tuberculosis and the Desirability of Resetting Cutoffs for Different Populations: A Retrospective Study.

Objectives The value of QuantiFERON-TB Gold In-Tube (QFT-GIT) in the diagnosis of TB varies by population, comorbidities, and other factors. In this study, we aimed to investigate factors that influence false-negative results of QFT-GIT test in the diagnosis of TB as well as the impact of different cutoffs on the diagnostic value. Methods A total of 3562 patients who underwent QFT-GIT tests at Shanghai Pulmonary Hospital were enrolled retrospectively between May 2016 and May 2017. False-negative and false-positive results were analyzed using different clinical stratifications. The optimal cutoff values were established under different clinical conditions. Results Positive QFT-GIT results greatly shortened the time taken to diagnose smear-negative TB. The factors of age, smear and culture results, site of TB, comorbidity with tumors, white blood cell count, neutrophil count, and CD4/CD8 ratio were significantly correlated with false-negative QFT-GIT results (p < 0.05). Personalized cutoff values were established according to different influencing factors. The results showed high consistency between the smear-negative and total populations. Conclusion QFT-GIT can facilitate the early diagnosis of smear-negative TB. The diagnostic performance of the QFT-GIT test in the diagnosis of active TB was shown to be affected by many clinical factors. Personalized cutoff values may have superior value in the identification of active tuberculosis under different conditions.

Efficacy of the Xpert Mycobacterium tuberculosis/rifampicin assay for diagnosing sputum-smear negative or sputum-scarce pulmonary tuberculosis in bronchoalveolar lavage fluid.

OBJECTIVE: To evaluate he diagnostic performance of the Xpert Mycobacterium tuberculosis/Rifampin (MTB/RIF) assay in bronchoalveolar lavage fluid (BALF). METHODS: We retrospectively reviewed the clinical data from 671 sputum-smear negative or sputum-scarce adult patients with suspected pulmonary tuberculosis (PTB) who had an Xpert MTB/RIF assay performed on BALF. The diagnostic performance of the Xpert MTB/RIF assay, smear microscopy (SM) and MTB culture was evaluated using MTB culture or final clinical diagnosis as the reference standard. RESULTS: Compared with MTB culture, the sensitivity and specificity were 87.8% and 72.7% for the Xpert MTB/RIF assay and 11.0% and 99.2% for SM, respectively. Compared with final diagnosis, diagnostic performance was 58.9% and 83.9% for the Xpert MTB/RIF assay, 5.0% and 98.3% for SM, and 43.3% and 100% for culture, for sensitivity and specificity respectively. The Xpert MTB/RIF assay had low specificity and high sensitivity. When very low results were re-evaluated and considered MTB-negative, the specificity increased significantly. The sensitivity remained higher than SM and was similar to that of culture. CONCLUSIONS: The Xpert MTB/RIF assay adds microbiologic evidence to clinical decisions; however, close attention should be paid to very low semi-quantitative positive results.

Characterization and identification of saikosaponins in crude extracts from three Bupleurum species using LC-ESI-MS.

A LC-diode array detection (DAD)-ESI-MS/MS method was established for the online characterization and identification of saikosaponins (SSs) from extracts of roots of Bupleurum scorzonerifolium Willd, B. marginatum var. stenophyllum and B. komarovianum. In ESI-MS/MS spectra of SSs, [M-H](- )ions were subjected to the cleavage of glycosidic bonds and produced Y type ions, which can be used to elucidate the structures of saccharide chains and aglycones. Fragmentation of aglycones provided mass information about their major substitutions. For three structural types of SSs, type III can be easily identified by their fragmentation behaviors; while type I and II often occur as isomers and they can be discriminated by their typical UV absorption data. The only sugar ring-cross cleavage corresponding to 76 Da took place at a furanose sugar moiety. As a result, more than 75 SSs, including eight novel compounds, were identified or tentatively characterized based on their UV and mass spectra and retention times. The approach established here allows a comprehensive analysis of the SSs in the genus of Bupleurum and will be helpful for quality control of the crude materials and their related preparations.