Uncovering the Bronchoalveolar Single-Cell Landscape of Patients With Pulmonary Tuberculosis With Human Immunodeficiency Virus Type 1 Coinfection.

BACKGROUND: Coinfection of human immunodeficiency virus type 1 (HIV-1) is the most significant risk factor for tuberculosis (TB). The immune responses of the lung are essential to restrict the growth of Mycobacterium tuberculosis and avoid the emergence of the disease. Nevertheless, there is still limited knowledge about the local immune response in people with HIV-1-TB coinfection. METHODS: We employed single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid from 9 individuals with HIV-1-TB coinfection and 10 with pulmonary TB. RESULTS: A total of 19 058 cells were grouped into 4 major cell types: myeloid cells, T/natural killer (NK) cells, B cells, and epithelial cells. The myeloid cells and T/NK cells were further divided into 10 and 11 subsets, respectively. The proportions of dendritic cell subsets, CD4+ T cells, and NK cells were lower in the HIV-1-TB coinfection group compared to the TB group, while the frequency of CD8+ T cells was higher. Additionally, we identified numerous differentially expressed genes between the CD4+ and CD8+ T-cell subsets between the 2 groups. CONCLUSIONS: HIV-1 infection not only affects the abundance of immune cells in the lungs but also alters their functions in patients with pulmonary TB.

Bronchoalveolar lavage fluid analysis in patients with checkpoint inhibitor pneumonitis.

BACKGROUND: Checkpoint inhibitor pneumonitis (CIP) is a relatively uncommon but potentially life-threatening immune-related adverse event (irAE). Lung biopsies have not been commonly performed for CIP patients. Bronchoalveolar lavage fluid (BALF) analysis is a useful diagnostic approach for interstitial lung disease. However, BALF features were inconsistent across different studies. METHODS: We retrospectively reviewed the medical records of 154 patients with pathologically confirmed malignancies and suffering from CIPs between July 2018 and December 2022. Patients who had bronchoalveolar lavage (BAL) data available were enrolled in our study. Patient clinical, laboratory, radiological and follow-up data were reviewed and analyzed. RESULTS: The BALF differential cell count and lymphocyte subset analysis were performed for 42 CIP patients. There were 32 males (76.2%). The mean age at diagnosis of CIP was 62.0 ± 10.4 (range: 31-78) years. The median time to onset of CIP was 98.5 days after the start of immunotherapy. There were 18 patients (42.9%) with low-grade CIPs and 24 patients (57.1%) with high-grade CIPs. The mean lymphocyte percentage was 36.7 ± 22.5%. There were 34 (81%) CIP patients with a lymphocytic cellular pattern. The median ratio of CD3+CD4+/CD3+CD8+ lymphocytes was 0.5 (0.3, 1.0). The ratio was less than 1.0 for 31 CIP patients (73.8%). However, there was no significant difference in the BALF features between patients with low-grade CIPs and those with high-grade CIPs. CONCLUSIONS: The CD3+CD8+ lymphocytosis pattern was the main inflammatory profile in the BALF of CIP patients in this cohort. Targeting CD3+CD8+ lymphocytes might be a treatment option for CIPs.

Protein-Protein interactive networks identified in bronchoalveolar lavage of severe compared to nonsevere asthma.

INTRODUCTION: Previous bronchoalveolar lavage fluid (BALF) proteomic analysis has evaluated limited numbers of subjects for only a few proteins of interest, which may differ between asthma and normal controls. Our objective was to examine a more comprehensive inflammatory biomarker panel in quantitative proteomic analysis for a large asthma cohort to identify molecular phenotypes distinguishing severe from nonsevere asthma. METHODS: Bronchoalveolar lavage fluid from 48 severe and 77 nonsevere adult asthma subjects were assessed for 75 inflammatory proteins, normalized to BALF total protein concentration. Validation of BALF differences was sought through equivalent protein analysis of autologous sputum. Subjects' data, stratified by asthma severity, were analysed by standard statistical tests, principal component analysis and 5 machine learning algorithms. RESULTS: The severe group had lower lung function and greater health care utilization. Significantly increased BALF proteins for severe asthma compared to nonsevere asthma were fibroblast growth factor 2 (FGF2), TGFα, IL1Ra, IL2, IL4, CCL8, CCL13 and CXCL7 and significantly decreased were platelet-derived growth factor a-a dimer (PDGFaa), vascular endothelial growth factor (VEGF), interleukin 5 (IL5), CCL17, CCL22, CXCL9 and CXCL10. Four protein differences were replicated in sputum. FGF2, PDGFaa and CXCL7 were independently identified by 5 machine learning algorithms as the most important variables for discriminating severe and nonsevere asthma. Increased and decreased proteins identified for the severe cluster showed significant protein-protein interactions for chemokine and cytokine signalling, growth factor activity, and eosinophil and neutrophil chemotaxis differing between subjects with severe and nonsevere asthma. CONCLUSION: These inflammatory protein results confirm altered airway remodelling and cytokine/chemokine activity recruiting leukocytes into the airways of severe compared to nonsevere asthma as important processes even in stable status.

Interferon-responsive neutrophils and macrophages extricate SARS-CoV-2 Omicron critical patients from the nasty fate of sepsis.

The SARS-CoV-2 Omicron variant is characterized by its high transmissibility, which has caused a worldwide epidemiological event. Yet, it turns ominous once the disease progression degenerates into severe pneumonia and sepsis, presenting a horrendous lethality. To elucidate the alveolar immune or inflammatory landscapes of Omicron critical-ill patients, we performed single-cell RNA-sequencing (scRNA-seq) of bronchoalveolar lavage fluid (BALF) from the patients with critical pneumonia caused by Omicron infection, and analyzed the correlation between the clinical severity scores and different immune cell subpopulations. In the BALF of Omicron critical patients, the alveolar violent myeloid inflammatory environment was determined. ISG15+ neutrophils and CXCL10+ macrophages, both expressed the interferon-stimulated genes (ISGs), were negatively correlated with clinical pulmonary infection score, while septic CST7+ neutrophils and inflammatory VCAN+ macrophages were positively correlated with sequential organ failure assessment. The percentages of ISG15+ neutrophils were associated with more protective alveolar epithelial cells, and may reshape CD4+ T cells to the exhaustive phenotype, thus preventing immune injuries. The CXCL10+ macrophages may promote plasmablast/plasma cell survival and activation as well as the production of specific antibodies. As compared to the previous BALF scRNA-seq data from SARS-CoV-2 wild-type/Alpha critical patients, the subsets of neutrophils and macrophages with pro-inflammatory and immunoregulatory features presented obvious distinctions, suggesting an immune disparity in Omicron variants. Overall, this study provides a BALF single-cell atlas of Omicron critical patients, and suggests that alveolar interferon-responsive neutrophils and macrophages may extricate SARS-CoV-2 Omicron critical patients from the nasty fate of sepsis.

The first case report: diagnosis and management of necrotizing fusobacterium lung abscess via BALF next-generation sequencing.

BACKGROUND: Fusobacterium necrophorum (F. necrophorum)-induced necrotizing pneumonia is a rare but severe pulmonary infection. Insufficient microbiological detection methods can lead to diagnostic difficulties. METHODS: We report a case of F. necrophorum lung abscess diagnosed by next-generation sequencing (NGS) of bronchoalveolar lavage fluid (BALF). RESULTS: BALF-NGS detected F. necrophorum, guiding subsequent targeted antibiotic therapy. With active drainage and metronidazole treatment, the patient's condition was effectively treated. CONCLUSION: BALF-NGS is a valuable tool for the rapid diagnosis of infections caused by difficult-to-culture bacteria. It played a decisive role in the early identification of F. necrophorum, enabling timely and targeted antibiotic intervention. Early diagnosis and appropriate treatment are crucial for the management of F. necrophorum pneumonia.

The etiological diagnostic value of metagenomic next-generation sequencing in suspected community-acquired pneumonia.

BACKGROUND: The emergence of metagenomic next-generation sequencing (mNGS) may provide a promising tool for early and comprehensive identification of the causative pathogen in community-acquired pneumonia (CAP). In this study, we aim to further evaluate the etiological diagnostic value of mNGS in suspected CAP. METHODS: A total of 555 bronchoalveolar lavage fluid (BALF) samples were collected for pathogen detection by mNGS from 541 patients with suspected CAP. The clinical value was assessed based on infection diagnosis and treatment guidance. The diagnostic performance for pathogen identification by mNGS and sputum culture and for tuberculosis (TB) by mNGS and X-pert MTB/RIF were compared. To evaluate the potential for treatment guidance, we analyzed the treatment regimen of patients with suspected CAP, including imaging changes of lung after empirical antibacterial therapy, intensified regimen, antifungal treatment, and a 1-year follow up for patients with unconfirmed diagnosis and non-improvement imaging after anti-infective treatment and patients with high suspicion of TB or NTM infection who were transferred to the Wuhan Pulmonary Hospital for further diagnosis and even anti-mycobacterium therapy. RESULTS: Of the 516 BALF samples that were analyzed by both mNGS and sputum culture, the positivity rate of mNGS was significantly higher than that of sputum culture (79.1% vs. 11.4%, P = 0.001). A total of 48 samples from patients with confirmed TB were analyzed by both mNGS and X-pert MTB/RIF, and the sensitivity of mNGS for the diagnosis of active TB was significantly lower than that of X-pert MTB/RIF (64.6% vs. 85.4%, P = 0.031). Of the 106 pathogen-negative cases, 48 were ultimately considered non-infectious diseases, with a negative predictive value of 45.3%. Of the 381 pathogen-positive cases, 311 were eventually diagnosed as CAP, with a positive predictive value of 81.6%. A total of 487 patients were included in the evaluation of the therapeutic effect, and 67.1% improved with initial empirical antibiotic treatment. Of the 163 patients in which bacteria were detected, 77.9% improved with antibacterial therapy; of the 85 patients in which fungi were detected, 12.9% achieved remission after antifungal therapy. CONCLUSIONS: Overall, mNGS had unique advantages in the detection of suspected CAP pathogens. However, mNGS was not superior to X-pert MTB/RIF for the diagnosis of TB. In addition, mNGS was not necessary as a routine test for all patients admitted with suspected CAP. Furthermore, when fungi are detected by mNGS, antifungal therapy should be cautious.

The correlation between the level of cellular classification in bronchoalveolar lavage fluid of children with severe Mycoplasma pneumoniae and clinical characteristics.

Mycoplasma pneumoniae pneumonia (MPP) is a common respiratory tract infection disease in children. To date, there have been few studies on the relationship between cytological changes in bronchoalveolar lavage fluid (BALF) and clinical features. The objective of this study is to investigate the correlation between changes in the proportion of cell classifications in BALF and the clinical features in children with severe MPP (SMPP). In total, the study included 64 children with SMPP requiring bronchoalveolar lavage who were admitted to our hospital between March and September 2022 (study group) and 11 children with bronchial foreign bodies without co-infection (control group), who were admitted during the same period. The proportion of cell classifications in BALF was determined by microscopic examination after performing Wright-Giemsa staining. Patients were grouped according to different clinical characteristics, and between-group comparisons were made regarding the variations in the proportion of cell classifications in BALF. The levels of blood routine neutrophil percentage (GRA%), C-reactive protein, D-dimer and lactate dehydrogenase in the study group were higher than those in the control group (P < 0.05). There were differences in the GRA% and macrophage percentage in the BALF between the two groups (P < 0.05). The GRA% and blood lymphocyte percentage were associated with pleural effusion. Multiple indicators correlated with extrapulmonary manifestations (P < 0.05). Moreover, the percentage of lymphocytes in the BALF correlated with pleural effusion, extrapulmonary manifestations and refractory MPP (RMPP) (P < 0.05). Logistic regression showed that BALF lymphocytes were protective factors for RMPP, while serum amyloid A and extrapulmonary manifestations were risk factors (P < 0.05). CONCLUSION: The BALF of children with SMPP is predominantly neutrophilic. A lower percentage of lymphocytes is related to a higher incidence of pleural effusion, extrapulmonary manifestations and progression to RMPP, as well as a longer length of hospitalisation. WHAT IS KNOWN: • Mycoplasma pneumonia in children is relatively common in clinical practice. Bronchoalveolar lavage (BAL) is a routine clinical procedure. WHAT IS NEW: However, there are relatively few studies focusing on the cytomorphological analysis of cells in BAL fluid.

Determination of the diagnostic accuracy of nanopore sequencing using bronchoalveolar lavage fluid samples from patients with sputum-scarce pulmonary tuberculosis.

PURPOSE: The early and efficient diagnosis of patients suspected of having pulmonary tuberculosis (PTB) remains challenging. This study aimed to evaluate the accuracy of nanopore sequencing for PTB diagnosis using bronchoalveolar lavage fluid (BALF) samples and compared it with other techniques such as acid-fast bacilli smear, culture, Xpert MTB/RIF, and CapitalBio Mycobacterium reverse transcription-polymerase chain reaction (MTB RT-PCR). METHODS: We retrospectively analyzed the clinical data of 195 patients with suspected PTB who were admitted to our hospital. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic curve (AUC) of these assays were calculated and compared. RESULTS: The overall sensitivity, specificity, PPV, NPV, and AUC of nanopore sequencing were 90.70%, 84.85%, 92.13%, 82.35%, and 0.88; those of acid-fast bacilli smear were 12.40%, 98.48%, 94.12%, 36.52%, and 0.55; those of culture were 36.43%, 100%, 100%, 44.59%, and 0.68; those of Xpert MTB/RIF were 41.09%, 100%, 100%, 46.48%, and 0.71; and those of CapitalBio MTB RT-PCR were 34.88%, 98.48%, 97.83%, 43.62%, and 0.67, respectively. CONCLUSION: The nanopore sequencing assay using BALF samples showed the best diagnostic accuracy for sputum-scarce PTB. Moreover, it can improve the clinical diagnosis of PTB.

Prognostic performance of Krebs von den Lungen-6, surfactant protein A, surfactant protein D levels in the serum and bronchoalveolar lavage fluid in chronic fibrosing interstitial pneumonia: a retrospective study.

BACKGROUND: The serum markers Krebs von den Lungen-6 (KL-6), surfactant protein A (SP-A), and surfactant protein D (SP-D) have been used for the diagnosis, differential diagnosis, and prognosis prediction of interstitial pneumonia. However, the significance of measuring the serum and bronchoalveolar lavage fluid (BALF) KL-6, SP-D, and SP-A levels in predicting the prognosis of chronic fibrosing interstitial pneumonia (CFIP), idiopathic pulmonary fibrosis, and idiopathic nonspecific interstitial pneumonia remains unclear. We aimed to clarify the significance of measuring the serum and BALF KL-6, SP-A, and SP-D levels in predicting the prognosis of patients with CFIP. METHODS: Among 173 patients who were diagnosed with CFIP between September 2008 and February 2021, 39 who underwent bronchoalveolar lavage were included in this study. Among these, patients experiencing an annual decrease in forced vital capacity (FVC) of ≥10% or those facing challenges in undergoing follow-up pulmonary function tests owing to significant deterioration in pulmonary function were categorized as the rapidly progress group. Conversely, individuals with an annual decrease in the FVC of <10% were classified into the slowly progress group. The serum and BALF KL-6, SP-D, and SP-A levels, as well as BALF/serum SP-D and SP-A ratios were compared between the two groups. RESULTS: Among the patients with CFIP, the BALF SP-D level (p=0.0111), BALF SP-A level (p<0.0010), BALF/serum SP-D ratio (p=0.0051), and BALF/serum SP-A ratio (p<0.0010) were significantly lower in the rapidly than in the slowly progress group (p<0.0010). The receiver operating characteristics analysis results demonstrated excellent performance for diagnosing patients with CFIP, with the BALF SP-D level (area under the curve [AUC], 0.7424), BALF SP-A level (AUC, 0.8842), BALF/serum SP-D ratio (AUC, 0.7673), and BALF/serum SP-A ratio (AUC, 0.8556). Moreover, the BALF SP-A level showed a notably superior CFIP diagnostic capability. Survival analysis using the Kaplan-Meier method revealed that patients with a BALF SP-A level of <1500 ng/mL and BALF/serum SP-A ratio of <15.0 had poor prognoses. CONCLUSIONS: Our results suggest that BALF SP-A measurement may be useful for predicting the prognosis in patients with CFIP.

Exploring the microbiota difference of bronchoalveolar lavage fluid between community-acquired pneumonia with or without COPD based on metagenomic sequencing: a retrospective study.

BACKGROUND: Community-acquired pneumonia (CAP) patients with chronic obstructive pulmonary disease (COPD) have higher disease severity and mortality compared to those without COPD. However, deep investigation into microbiome distribution of lower respiratory tract of CAP with or without COPD was unknown. METHODS: So we used metagenomic next generation sequencing (mNGS) to explore the microbiome differences between the two groups. RESULTS: Thirty-six CAP without COPD and 11 CAP with COPD cases were retrieved. Bronchoalveolar lavage fluid (BALF) was collected and analyzed using untargeted mNGS and bioinformatic analysis. mNGS revealed that CAP with COPD group was abundant with Streptococcus, Prevotella, Bordetella at genus level and Cutibacterium acnes, Rothia mucilaginosa, Bordetella genomosp. 6 at species level. While CAP without COPD group was abundant with Ralstonia, Prevotella, Streptococcus at genus level and Ralstonia pickettii, Rothia mucilaginosa, Prevotella melaninogenica at species level. Meanwhile, both alpha and beta microbiome diversity was similar between groups. Linear discriminant analysis found that pa-raburkholderia, corynebacterium tuberculostearicum and staphylococcus hominis were more enriched in CAP without COPD group while the abundance of streptococcus intermedius, streptococcus constellatus, streptococcus milleri, fusarium was higher in CAP with COPD group. CONCLUSIONS: These findings revealed that concomitant COPD have an mild impact on lower airway microbiome of CAP patients.

Metagenomic next-generation sequencing promotes diagnosis and treatment of Pneumocystis jirovecii pneumonia in non-HIV infected children: a retrospective study.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) excels in diagnosis of infection pathogens. We aimed to evaluate the performance of mNGS for the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in non-HIV infected children. METHODS: Totally 36 PJP children and 61 non-PJP children admitted to the pediatric intensive care unit from March 2018 to December 2021 were retrospectively enrolled. Clinical features of PJP children were summarized. 1,3-ß-D glucan (BDG) test and bronchoalveolar lavage fluid (BALF) mNGS were used for evaluation of PJP diagnostic performance. Antimicrobial management modifications for PJP children after the mNGS results were also reviewed. RESULTS: Pneumocystis jirovecii was detected in all PJP children by mNGS (36/36), and the sensitivity of mNGS was 100% (95% confidence interval [CI]: 90.26-100%). The sensitivity of BDG was 57.58% (95% CI: 39.22-74.52%). Of the 26 (72.2%) PJP patients with mixed infection, twenty-four (66.7%) were detected by BALF-mNGS. Thirteen patients (36.1%) had their antimicrobial management adjusted according to the mNGS results. Thirty-six PJP children included 17 (47.2%) primary immunodeficiency and 19 (52.8%) secondary immunodeficiency, of whom 19 (52.8%) survived and 17 (47.2%) died. Compared to survival subgroup, non-survival subgroup had a higher rate of primary immunodeficiency (64.7% vs. 31.6%, P = 0.047), younger age (7 months vs. 39 months, P = 0.011), lower body weight (8.0 kg vs. 12.0 kg, P = 0.022), and lower T lymphocyte counts. CONCLUSIONS: The mortality rate of PJP in immunosuppressed children without HIV infection is high and early diagnosis is challenging. BALF-mNGS could help identify PJP and guide clinical management.

Clinical utility of metagenomic next-generation sequencing on bronchoalveolar lavage fluid in diagnosis of lower respiratory tract infections.

BACKGROUND: In this study, we aimed to evaluate the clinical utility of Metagenomic Next-Generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) in diagnosis of Lower Respiratory Tract Infections (LRTIs). METHODS: In this study, we retrospectively analyzed 186 hospitalized patients who were suspected with LRTIs and performed mNGS (DNA) test of BALF simultaneously at The Fifth Affiliated Hospital of Sun Yat-Sen University from March 2023 to August 2023. Suspected LRTI was based on LRTI related clinical manifestations or imaging examination. Among them, 155 patients had undergone conventional culture and mNGS (DNA) testing simultaneously. Finally, 138 cases (89.03%,138/155) were diagnosed as LRTI and 17 cases (10.97%,17/155) were diagnosed as non-LRTI. Both detecting rate and diagnostic efficacy of mNGS and conventional culture were compared. RESULTS: The positive detection rates of pathogens between mNGS and conventional culture were significant different (81.29% VS 39.35%, P < 0.05). Compared with paired conventional culture result, the sensitivity of mNGS in diagnosis of LRTIs was more superior (88.41% VS 43.48%; P < 0.05), the specificity was opposite (76.47% VS 94.12%; P > 0.05). Furthermore, 77.54% and 35.51% of LRTI cases were being etiologically diagnosed by mNGS and culture respectively. Importantly, mNGS directly led to a change of treatment regimen in 58 (37.42%) cases, including antibiotic adjustment (29.68%) and ruling out active infection (7.74%). Moreover, treatment regimen remained unchanged in 97 (62.58%) cases, considering the current antibiotic therapy already covered the detected pathogens (36.13%) or empirical treatment was effective (11.61%). CONCLUSIONS: mNGS can identify a wide range of pathogens in LRTIs, with improved sensitivity and being more superior at diagnosing LRTIs etiologically. mNGS has the potential to enhance clinical outcomes by optimizing the treatment regimens.

Value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in acute exacerbation of fibrosing interstitial lung disease: an individualized treatment protocol based on microbiological evidence.

BACKGROUND: Acute exacerbation of fibrosing interstitial lung diseases (AE-ILD) is a serious life-threatening event per year. Methylprednisolone and/or immunosuppressive agents (ISA) are a mainstay in any regimen, under the premise that pulmonary infection has been promptly identified and controlled. We investigated the value of bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) on the treatment adjustment of AE-ILD. METHODS: We conducted a cross-sectional observational study. All data were collected prospectively and retrospectively analyzed. We included fifty-six patients with AE-ILD and nineteen stable ILD who underwent BALF mNGS at the beginning of admission. RESULTS: Patients with a variety of ILD classification were included. Connective-tissue disease related ILD (CTD-ILD) occupy the most common underlying non-idiopathic pulmonary fibrosis (non-IPF). The infection-triggered AE accounted for 39.29%, with the majority of cases being mixed infections. The microorganisms load in the AE-ILD group was significantly higher. After adjusted by mNGS, the therapy coverage number of pathogens was significantly higher compared to the initial treatment (p < 0.001). After treatment, the GGO score and the consolidation score were significantly lower during follow up in survivors (1.57 ± 0.53 vs. 2.38 ± 0.83 with p < 0.001, 1.11 ± 0.24 vs. 1.49 ± 0.47 with p < 0.001, respectively). Some detected microorganisms, such as Tropheryma whipplei, Mycobacterium, Aspergillus, and mixed infections were difficult to be fully covered by empirical medication. BALF mNGS was also very helpful for excluding infections and early administration of methylprednisolone and/or ISA. CONCLUSIONS: mNGS has been shown to be a useful tool to determine pathogens in patients with AE-ILD, the results should be fully analyzed. The comprehensive treatment protocol based on mNGS has been shown crucial in AE-ILD patients.

Metabolic landscape dysregulation in bronchoalveolar lavage fluid of checkpoint inhibitor pneumonitis.

BACKGROUND: Checkpoint inhibitor pneumonitis (CIP) is a potentially fatal adverse event resulting from immunotherapy in patients with malignant tumors. However, the pathogenesis of CIP remains poorly understood. METHODS: We collected bronchoalveolar lavage fluid (BALF) from cohorts of patients with CIP, new-onset lung cancer (LC), and idiopathic pulmonary fibrosis (IPF). Non-targeted metabolomics analysis was conducted to analyze metabolic signatures. Flow cytometry was used to evaluate immune cell subsets. RESULTS: Lymphocytes were predominant in the BALF of patients with CIP. A total of 903 metabolites were identified, among which lipid compounds were the most abundant. In a comparison between patients with CIP and LC, enrichment analysis of the altered metabolites showed suppressed amino sugar metabolism, and spermidine and spermine biosynthesis in the CIP group. Metabolism of alpha linolenic acid, linoleic acid, and their fatty acid derivatives was enriched in the CIP group relative to the IPF group. The twelve metabolites found to be enriched in the CIP group were positively correlated with the proportion of CD8+ T cells. One cluster of BALF metabolites, 57.14% of which were lipid molecules, was inversely correlated with the proportion of natural killer cells. CONCLUSIONS: In this study, the metabolomic landscape of BALF in patients with CIP was determined. We elucidated suppressed tumor metabolic signatures, enhanced pulmonary inflammatory signaling, and the characteristics of responsible immune cells, which helps to understand the pathogenesis of CIP.

Coinfection with SARS-CoV-2 and Influenza A Virus Increases Disease Severity and Impairs Neutralizing Antibody and CD4+ T Cell Responses.

Given the current coronavirus disease 2019 (COVID-19) pandemic, coinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) is a major concern for public health. However, the immunopathogenic events occurring with coinfections of SARS-CoV-2 and IAV remain unclear. Here, we report the pathogenic and immunological consequences of SARS-CoV-2 and IAV H1N1 coinfection in the K18-hACE2 transgenic mouse model. Compared with a single infection with SARS-CoV-2 or IAV, coinfections not only prolonged the primary virus infection period but also increased immune cell infiltration and inflammatory cytokine levels in bronchoalveolar lavage fluid leading to severe pneumonia and lung damage. Moreover, coinfections caused severe lymphopenia in peripheral blood, resulting in reduced total IgG, neutralizing antibody titers, and CD4+ T cell responses against each virus. This study sheds light on the immunopathogenesis of SARS-CoV-2 and IAV coinfection, which may guide the development of effective therapeutic strategies for the treatment of patients coinfected with these viruses. IMPORTANCE The cocirculation of influenza virus merging with the COVID-19 pandemic raises a potentially severe threat to public health. Recently, increasing numbers of SARS-CoV-2 and influenza virus coinfection have been reported from many countries. It is a worrisome issue that SARS-CoV-2 coinfection with other pathogens may worsen the clinical outcome and severity of COVID-19 and increase fatality. Here, we evaluated SARS-CoV-2 and IAV coinfection using the K18-hACE2 mouse model. Coinfected mice exhibited increased mortality with prolonged IAV shedding. Furthermore, coinfected mice showed a higher level of cytokines and chemokines than a single infection condition. Interestingly, our data show that coinfected mice showed significantly fewer virus-specific and neutralizing antibodies than the mice with a single infection. Overall, this study suggests that coinfection aggravates viral pathology by impaired neutralizing antibody response.

Characterizing the lavage and serum cytokine profiles of interstitial pneumonia with autoimmune features and their implications for progressive fibrosis.

OBJECTIVE: To explore whether cytokines could be potential biomarkers to predict the occurrence of progressive fibrosis (PF) phenotype among interstitial pneumonia with autoimmune features (IPAF) patients. METHODS: This study prospectively collected 51 IPAF and 15 Idiopathic Pulmonary Fibrosis (IPF) patients who were diagnosed at First Affiliated Hospital of Guangzhou Medical University from July 2020 to June 2021. All IPAF patients were followed up for one year to assess the development of PF phenotype. Paired Broncho Alveolar Lavage Fluid (BALF) and serum samples were collected at enrolment and analyzed for differences in 39 cytokines expression. Principal component analysis (PCA) and cluster analysis were conducted to identify a high-risk subgroup of IPAF patients for developing the PF phenotype. Finally, cytokine differences were compared between subgroups to identify potential biomarkers for PF-IPAF occurrence. RESULTS: According to the PCA analysis, 81.25% of PF-IPAF patients share overlapped BALF cytokine profiles with IPF. Cluster analysis indicated IPAF patients in subtype 2 had a higher risk to develop PF phenotype within one year (P = 0.048), characterized by higher levels of CCL2, CXCL12 and lower lymphocyte proportion (LYM%) in BALF. Elevated levels of BALF CCL2 (>299.16 pg./ml) or CXCL12 (>600.115 pg./ml) were associated with a significantly higher risk of developing PF phenotype within one year follow-up period (P = 0.009, 0.001). CONCLUSION: PF-IPAF phenotype exhibits similar inflammatory cytokine profiles to IPF. Cytokine CCL2, CXCL12, and LYM% in BALF serving as potential biomarkers for predicting the PF phenotype in IPAF patients. CLINICAL TRIAL REGISTRATION: Register: Qian Han, Website: http://www.chictr.org.cn/showproj.aspx?proj=61619, Registration number: ChiCTR2000040998.

An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples.

BACKGROUND: Clinical bronchoalveolar lavage fluid (BALF) samples are rich in biomolecules, including proteins, and useful for molecular studies of lung health and disease. However, mass spectrometry (MS)-based proteomic analysis of BALF is challenged by the dynamic range of protein abundance, and potential for interfering contaminants. A robust, MS-based proteomics compatible sample preparation workflow for BALF samples, including those of small and large volume, would be useful for many researchers. RESULTS: We have developed a workflow that combines high abundance protein depletion, protein trapping, clean-up, and in-situ tryptic digestion, that is compatible with either qualitative or quantitative MS-based proteomic analysis. The workflow includes a value-added collection of endogenous peptides for peptidomic analysis of BALF samples, if desired, as well as amenability to offline semi-preparative or microscale fractionation of complex peptide mixtures prior to LC-MS/MS analysis, for increased depth of analysis. We demonstrate the effectiveness of this workflow on BALF samples collected from COPD patients, including for smaller sample volumes of 1-5 mL that are commonly available from the clinic. We also demonstrate the repeatability of the workflow as an indicator of its utility for quantitative proteomic studies. CONCLUSIONS: Overall, our described workflow consistently provided high quality proteins and tryptic peptides for MS analysis. It should enable researchers to apply MS-based proteomics to a wide-variety of studies focused on BALF clinical specimens.

Mycobacterium tuberculosis-specific memory T cells in bronchoalveolar lavage of patients with pulmonary tuberculosis.

BACKGROUND: Mycobacterium tuberculosis(MTB) most often infects the lungs and results in pulmonary tuberculosis(TB). MTB-specific memory T cells are able to respond quickly against antigens and help reduce the burden of pulmonary bacteria. The characteristics, function and chemotaxis axis of memory T cells in the lung remain unclear. The current study aimed to clarify the classification, function and recruitment of local antigen-specific memory T cells in the lung and the periphery blood of patients with pulmonary TB. METHODS: A total of 85 patients with active pulmonary TB were included in the study. Bronchoalveolar lavage fluid (BALF) and Peripheral blood were collected for further detection. The cell-surface markers and intracellular staining of memory T cell subtypes were measured by flow cytometry. The level of CXCL9, CXCL10 and CXCL11 in Bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells (PBMC) were measured by Real-time PCR. RESULTS: The ratio of effective Memory T cells (TEM) were the highest in BALF of patients with pulmonary TB. In patients, CXCR3 and its ligands was increased in memory T cells of BALF compared with PBMC. IFN-γ+TNF-α+ effective Memory T cells and central memory T cells from BALF were increased after antigen stimulation. CXCR3 was higher in IFN-γ+ compared with IFN-γ- in CD4+ TCM and TEM from BALF of patients. Compared with PBMC, the PD-1 levels of terminal effector memory RA+(TEMRA) and TEM cells in CD4+ memory T cells of BALF were significantly increased. In addition, PD-1 was increased in IFN-γ+ compared with IFN-γ- in CD4+TEM from BALF of patients. There was no difference in Treg ratio between PBMC and BALF of TB patients. CONCLUSIONS: The CXCL9/CXCL11-CXCR3 axis may participate in the chemotaxis of memory T cells from the peripheral to lung. CD4+TEM and TEMRA in BALF may have exhausted, especially the cytokine producing TEM. Our study clarified the characteristics of antigen-specific memory T cells in local lung and may have impact on strategies of therapy and vaccine.

Curtanaerobium respiraculi gen. nov., sp. nov., a novel anaerobic bacterium isolated from human bronchoalveolar lavage fluid.

Two related anaerobic strains, designated as SWB101512T and SWB19611, were isolated from the bronchoalveolar lavage fluid of two lung cancer patients. Cells were Gram-stain-positive, non-motile and non-spore-forming. Growth could be observed at 26-45 °C (optimum, 37 °C), pH 5.0-8.5 (optimum, pH 7.0) and with 0.5-2.0 % (v/w) NaCl (optimum, 1.0%). The 16S rRNA gene sequences of SWB101512T and SWB19611 showed the highest similarities to Denitrobacterium detoxificans DSM 21843T (91.1 and 91.3 %, respectively). The phylogenetic tree based on the 16S rRNA gene sequences and the core genome sequences demonstrated that the two strains clustered together and formed a distinct lineage within the family Eggerthellaceae. The DNA G+C contents of strains SWB101512T and SWB19611 were 62.0 and 61.9 mol%, respectively. The predominant cellular fatty acids of strains SWB101512T and SWB19611 were C16 : 0 DMA (27.8 and 28.8 %, respectively). The respiratory menaquinone in both strains was menaquinone 6 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, three glycolipids and three unidentified lipids. Based on evidence from phenotypic, chemotaxonomic and genomic analyses, a new genus and species belonging to the family Eggerthellaceae, named Curtanaerobium respiraculi gen. nov., sp. nov. is proposed. The type strain is SWB101512T (=GDMCC 1.2991T=JCM 35330T).

Comparison of endotracheal aspirate and bronchoalveolar lavage fluid metagenomic next-generation sequencing in severe pneumonia: a nested, matched case-control study.

OBJECTIVES: To compare clinical outcomes in patients with severe pneumonia according to the diagnostic strategy used. METHODS: In this retrospective, nested, case-control study, patients with severe pneumonia who had undergone endotracheal aspirate (ETA) metagenomic next-generation sequencing of (mNGS) testing (n = 53) were matched at a ratio of 1 to 2 (n = 106) by sex, age, underlying diseases, immune status, disease severity scores, and type of pneumonia with patients who had undergone bronchoalveolar lavage fluid (BALF) mNGS. The microbiological characteristics and patient's prognosis of the two groups were compared. RESULTS: An overall comparison between the two groups showed no significant differences in bacterial, fungal, viral, or mixed infections. However, subgroup analysis of 18 patients who received paired ETA and BALF mNGS showed a complete agreement rate for the two specimens of 33.3%. There were more cases for whom targeted treatment was initiated (36.79% vs. 22.64%; P = 0.043) and fewer cases who received no clinical benefit after mNGS (5.66% vs. 15.09%; P = 0.048) in the BALF group. The pneumonia improvement rate in the BALF group was significantly higher than in the ETA group (73.58% vs. 87.74%, P = 0.024). However, there were no significant differences in ICU mortality or 28-day mortality. CONCLUSIONS: We do not recommend using ETA mNGS as the first-choice method for analyzing airway pathogenic specimens from severe pneumonia patients.