Search Results (89)

Conventional diagnostic methods (CDMs) for lower respiratory infections (LRIs) have limitations in detecting causative pathogens. This study evaluates the utility of shotgun metagenomic sequencing (SMS) as a complementary diagnostic tool using bronchoalveolar lavage (BAL) fluid. Sixteen BAL fluid samples from pneumonia patients with positive CDM results—including bacterial/fungal cultures; PCR for Mycobacterium tuberculosis or cytomegalovirus; and the BioFire^® FilmArray^® Pneumonia Panel (BioFire Diagnostics LLC, Salt Lake City, UT, USA)—underwent 10 Gb SMS on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Reads were aligned to the NCBI RefSeq database; with fungal identification further supported by internal transcribed spacer (ITS) analysis. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database. Microbial reads accounted for 0.00002–0.04971% per sample. SMS detected corresponding bacteria in 63% of cases, increasing to 69% when subdominant taxa were included. Fungal reads were low; however, Candida species were identified in four samples via ITS. No viral reads were detected. ARGs meeting perfect match criteria were found in two cases. This is the first real-world study comparing SMS with CDMs, including semiquantitative PCR, in BAL fluid for LRI. SMS shows promise as a supplementary diagnostic method, with further research needed to optimize its performance and cost-effectiveness. Full article

Background: Bronchoalveolar lavage fluid (BALF) analysis is essential for the accurate diagnosis and management of interstitial lung diseases (ILDs). Despite established guidelines, variability in sample handling may affect diagnostic accuracy. This study aimed to evaluate how different storage conditions impact BALF cell counts and differentials to guide optimal sample handling practices. Methods: Forty patients who underwent BAL at Chiba University Hospital from June to December 2024 were included. BALF samples were allocated into five groups based on processing conditions: immediate analysis within 1 h, storage at either at 4 °C or room temperature (RT) for 6 h, or storage at 4 °C or RT for 24 h. Total cell counts (TCC) and differential counts were measured and compared among conditions. Results: TCC remained stable over 24 h at both 4 °C (p = 0.86) and RT (p = 0.90). Similarly, the percentages of eosinophils, lymphocytes, and macrophages did not significantly change at either temperature (all p > 0.05). Notably, neutrophil percentages showed a significant decline over time under both storage conditions—at 4 °C (p = 0.02) and at room temperature (p < 0.01). Post hoc tests revealed a notable decreasing trend at 6 h and significant reductions by 24 h at 4 °C (p = 0.09 and p = 0.02, respectively), and significant decreases at both 6 and 24 h at RT (p = 0.01, <0.01). Conclusions: Among the various cell types in BALF, neutrophil proportions are particularly susceptible to storage conditions, showing a significant decline over time—especially at room temperature—while other cell types remain stable for up to 24 h. Therefore, prompt processing or appropriate refrigeration of BALF is essential to ensure reliable cytological analysis and accurate clinical interpretation. Full article

Background: Invasive aspergillosis (IA) is a life-threatening fungal infection that primarily affects immunocompromised individuals and has high morbidity and mortality rates, necessitating timely diagnosis and treatment. This study aimed to evaluate the prognostic utility of serum and bronchoalveolar lavage (BAL) fluid galactomannan levels, as well as galactomannan kinetics, in patients with IA. Methods: We retrospectively reviewed the medical records of patients who were diagnosed with proven or probable IA from March 2016 to April 2024 at a tertiary cancer center. The collected data included patient characteristics, baseline and peak galactomannan levels in serum and BAL fluid, galactomannan trends, and clinical outcomes. Subgroup analyses were performed to assess the prognostic value of dual-source galactomannan positivity (positive serum and BAL fluid galactomannan levels). Results: Elevated baseline serum galactomannan levels independently predicted treatment non-response (p = 0.039) and 12-week all-cause mortality (p < 0.001). Peak serum and BAL fluid galactomannan levels were strongly associated with poor clinical outcomes (p < 0.01). Compared to single-source galactomannan positivity, dual-source galactomannan positivity was linked to reduced treatment response (22% vs. 43%, p = 0.01) and higher IA-attributable mortality (52% vs. 27%, p = 0.002). Patients with neutropenia had poorer outcomes compared to patients without neutropenia, but neutrophil recovery dramatically improved survival (25% vs. 69% mortality, p < 0.0001). Early galactomannan kinetics and malignancy type had limited prognostic value. Conclusions: Our findings highlight the potential role of galactomannan as a key biomarker for early prognostication for IA. The strong association between galactomannan levels and clinical outcomes suggests its utility in identifying high-risk patients who may benefit from more aggressive management. Further studies are needed to introduce a nuanced and context-specific use of galactomannan into clinical practice and assess its role as a prognostic biomarker. Full article

Objective: To investigate the effect of Lipopolysaccharide (LPS)-induced acute lung injury (ALI) on the pulmonary pharmacokinetics (PK) of a systemically administered antibody in mice. Method: The PK of a non-target-binding antibody was evaluated in healthy mice and mice with intratracheal instillation of 5 mg/kg LPS. The plasma, bronchoalveolar lavage (BAL), trachea, bronchi, and lung homogenate PK of the antibody were measured following intravenous administration of 5 mg/kg antibody dose. Noncompartmental analysis was performed to determine AUC values. Antibody concentrations in all biological matrices were quantified using qualified ELISA. The effect of ALI on BAL albumin and total protein concentrations was also determined. BAL protein concentrations were corrected for dilution using plasma urea concentrations. Results: Intratracheal instillation of LPS and the resultant ALI led to ~2–4-fold higher concentrations of albumin and proteins in the BAL. LPS-induced ALI also notably altered the pulmonary PK of the antibody. The effect of ALI on the antibody PK was time and tissue dependent. The trachea and bronchi showed ~1.7-fold and ~1.4-fold lower antibody exposure compared with the control group, but the BAL fluid exhibited ~4-fold increase in antibody exposure following LPS treatment. Most noticeable changes in antibody PK occurred 24 h after LPS administration, and the effect was temporary for the bronchi and trachea. However, the changes in lung homogenate and, more notably, in BAL persisted until the end of the experiment. Thus, our investigation suggests that due to the acute nature of ALI-induced pathophysiology and the changing severity of the disease, the dose and timing of antibody administration following ALI may need to be optimized based on the target site of action (e.g., bronchi, trachea, BAL, lung parenchyma, etc.) to maximize the therapeutic effect of the antibody. Conclusions: ALI may significantly affect pulmonary PK of systemically administered antibodies. Changes caused by ALI are time and tissue dependent, and hence, the timing and dose of antibody following ALI may need to be optimized to maximize the therapeutic effect of the antibody at the site of action. Full article

Background: Inflammation plays a crucial role in lung cancer recurrence after surgery. This study aims to investigate the relationship between lung cancer recurrence and perioperative inflammatory status, assessed in both blood and bronchoalveolar lavage (BAL) fluid. Methods: We conducted a retrospective cohort study analyzing clinical variables, blood cytokine levels, and BAL fluid from lung cancer patients who underwent surgery. Logistic regression models were employed to predict recurrence. Results: Among 93 patients, 41.9% experienced recurrence within ten years. The logistic regression model identified vital status, tumor stage, and type of surgery as significant predictors of recurrence. Postoperatively, pro-inflammatory cytokines were elevated, particularly in patients who experienced recurrence. Higher levels of TNF-α in BAL fluid and increased IL-6 in blood correlated with recurrence. Additionally, metalloproteinases in BAL fluid exhibited distinct associations: MMP-2 was identified as a risk factor, whereas MMP-9 appeared to have a protective role. A multivariate model integrating clinical variables and inflammatory biomarkers significantly improved predictive accuracy (p < 0.0001). Discussion: Combining inflammatory biomarkers with clinical variables enhances the prediction of lung cancer recurrence after surgery. Understanding the dynamics of these biomarkers may facilitate early detection and enable more personalized treatment strategies. Full article

Integrin β4 (ITGB4) mediates lung endothelial cell (EC) inflammation attenuated by simvastatin, an HMG CoA-reductase inhibitor. The cytoplasmic domain of ITGB4 is predicted to bind kindlin-2. Kindlin-2 expression is mediated by SMURF1, an E3 ubiquitin ligase that promotes kindlin-2 ubiquitination and degradation. We hypothesized that increased kindlin-2 expression via the inhibition of SMURF1 mediates EC inflammatory responses relevant to acute lung injury (ALI). To investigate this, human lung ECs were treated with simvastatin (5 µM, 16 h) prior to the immunoprecipitation of kindlin-2 and Western blotting for ITGB4. Next, ECs were treated with a SMURF1 inhibitor, A01, and increased kindlin-2 expression was confirmed. In assays of barrier function, kindlin-2 was silenced (siRNA) in ECs prior to thrombin and measurements of transendothelial resistance (TER) and FITC-dextran transwell flux. Repeat assessments of barrier function were performed in A01-treated ECs. Finally, mice were pretreated with A01 prior to LPS; bronchoalveolar lavage (BAL) fluid was collected, and their lungs were used for histology. Simvastatin increased ITGB4:kindlin-2 association, while A01 increased kindlin-2 expression. Thrombin-induced EC barrier disruption was both increased after kindlin-2 silencing and decreased by A01. Finally, murine ALI was significantly attenuated by A01. Our findings suggest that the augmentation of kindlin-2 may serve as a novel ALI therapeutic strategy. Full article

Diffuse interstitial lung diseases (ILD) include conditions with identifiable causes such as chronic eosinophilic pneumonia (CEP), sarcoidosis (SAR), chronic hypersensitivity pneumonitis (CHP), and connective tissue disease-associated interstitial pneumonia (CTD), as well as idiopathic interstitial pneumonia (IIP) of unknown origin. In non-IIP diffuse lung diseases, bronchoalveolar lavage (BAL) fluid appearance is diagnostic. This study examines lymphocyte subsets in BAL fluid and peripheral blood of 56 patients with diffuse ILD, excluding idiopathic pulmonary fibrosis (IPF), who underwent BAL for diagnostic purposes. Patients were classified into CEP, SAR, CHP, CTD, and IIP groups, and clinical data, BAL cell analysis, and peripheral blood mononuclear cell analysis were compared. Eosinophils and type 3 innate lymphocytes (ILC3s) were significantly increased in the BAL fluid of the CEP group. Receiver operating characteristic curve analysis identified eosinophils ≥ 8% in BAL cells and ILC3s ≥ 0.0176% in the BAL lymphocyte fraction as thresholds distinguishing CEP. SAR patients exhibited significantly elevated CD4/CD8 ratios in the BAL fluid, with a ratio of 3.95 or higher and type 1 innate lymphoid cell frequency ≥ 0.254% as differentiation markers. High Th1 cell frequency (≥17.4%) in BAL lymphocytes in IIP, elevated serum KL-6 (≥2081 U/mL) and SP-D (≥261 ng/mL) in CHP, and increased BAL neutrophils (≥2.0%) or a low CD4/CD8 ratio (≤1.2) in CTD serve as distinguishing markers for each ILD. Excluding CEP and SAR, CD4⁺ T cell frequencies, including Th1, Th17, and Treg cells in peripheral blood, may differentiate IIP, CHP, and CTD. Full article

Grape seed procyanidin extract (GSE) is widely used to promote cardiovascular health and has purported anti-inflammatory properties. Chronic inflammation in the lungs caused by environmental toxins such as tobacco smoking plays a pivotal role in lung cancer development. In a modified phase I lung cancer chemoprevention study conducted in heavy active and former smokers using leucoselect phytosome (LP), a standardized grape seed procyanidin extract complexed with soy phospholipids to enhance bioavailability, three months of LP treatment favorably modulated a variety of surrogate endpoint biomarkers, including markers of cell proliferation. In this correlative study, we further analyzed the effects of LP on cytochrome P450 3A4 (CYP3A4) activities by comparing the endogenous conversions of cortisol and cortisone to 6-beta-hydroxycortisol and 6-beta-hydroxycortisone, respectively, before and after LP treatment and the anti-inflammatory effects of LP in the lung microenvironment of these participants by comparing a profile of inflammatory cytokines and chemokines in matched pre- and post-treatment bronchoalveolar lavage (BAL) fluids. LP treatment did not significantly alter CYP3A4 activity, and three months of LP treatment significantly decreased tumor necrosis factor (TNF), C-C Motif Chemokine Ligand 3 (CCL3) and granzyme B in BAL fluids. Furthermore, post-LP-treatment BAL fluids significantly reduced migration/invasion of various human lung neoplastic cells in vitro. Our findings support the anti-inflammatory effects of GSE/LP in the lung microenvironment and its potential utility for reducing cancerizing forces, as well as driving forces for other common respiratory diseases such as chronic obstructive pulmonary disease and asthma, in the lungs of heavy former and active smokers. Full article

Background/Objectives: Airborne exogenous antigen inhalation can induce neutrophil infiltration of the airways, while eosinophils migrate to the airways in allergic airway inflammation. During a bacterial infection, Th2-associated cytokine IL-4, by binding to the IL-4 receptor (IL-4R), can suppress neutrophil recruitment to the site of inflammation. In the present study, we estimated whether the IL-4-dependent suppression of neutrophil recruitment contributed to the development of an immune response in asthma. Methods: Using a mouse model of Aspergillus fumigatus extract-induced allergic airway inflammation, we investigated the proportions of eosinophils and neutrophils in blood, lungs, and bone marrow over time. Bronchoalveolar lavage (BAL) fluid cytokine (including IL-4) levels and the proportions of bone marrow IL-4Rα (CD124)-expressing neutrophils were estimated. Results: We identified skewing from the neutrophil- to eosinophil-mediated immune response in the blood after five extract applications. At this point, the BAL fluid IL-4 level was not elevated, while IL-12p40 and CXCL1 levels were considerably increased. At the early stage of allergic airway inflammation, the proportions of neutrophils expressing CD124 and circulating neutrophils expressing CXCR2 (CD182) were significantly increased. Upon inflammation progression, the former remained elevated, but the latter significantly decreased. Conclusions: Thus, in allergic airway inflammation, bone marrow neutrophils become insensible to the attractive chemokine CXCL1 signals and susceptible to IL-4 effects. Full article

To determine the etiological agents responsible for acute pneumonia in puppies in China, this study utilized bronchoalveolar lavage (BAL) fluid extraction to enable the isolation, culture, biochemical identification, and 16S rRNA PCR amplification of the pathogens. Following preliminary identification, the pathogens underwent analysis for antibiotic resistance phenotypes and resistance genes. Additionally, the study examined the presence of virulence genes, conducted multilocus sequence typing (MLST), and performed whole-genome sequencing (WGS). The findings revealed that all four isolated pathogens were characterized as extraintestinal pathogenic Escherichia coli (ExPEC). The examined ExPEC strains demonstrated resistance to cephalosporins, tetracyclines, and penicillins, while remaining susceptible to aminoglycosides, beta-lactamase inhibitors, carbapenems, chloramphenicols, and sulfonamides. An analysis of virulence genes identified the presence of eight genes, namely CNF-I, fyuA, fimC, papC, ompA, fimH, irp2, and iroN, which are implicated in their invasiveness and potential to inflict tissue damage. The MLST analysis revealed that all ExPEC strains were classified under either sequence type ST131 (Achtman database) or ST43 (Pasteur database). The study further determined that these strains were absent in the kennel’s drinking water source, thereby ruling out water contamination as a potential factor in the emergence of ST131-type ExPEC. This study offers a theoretical framework and empirical evidence for elucidating the potential pathogenic mechanisms and clinical therapeutic strategies of ExPEC in the etiology of acute pneumonia in puppies. Full article

Background and Objectives: Ventilator-associated pneumonia (VAP) is a common complication in critically ill patients receiving mechanical ventilation. The incidence rates of VAP vary, and it poses significant challenges due to microbial resistance and the potential for adverse outcomes. This study aims to explore the microbial profile of VAP and evaluate the utility of biomarkers and illness severity scores in predicting survival. Materials and Methods: A retrospective cohort study was conducted involving 130 patients diagnosed with VAP. Microbial analysis of bronchoalveolar lavage (BAL) fluid, as well as measurements of C-reactive protein (CRP) and procalcitonin (PCT) levels, were performed. Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were calculated to assess illness severity. Statistical analyses were conducted to determine correlations and associations. Results: The study revealed that Klebsiella pneumoniae (K. pneumoniae) (50.7%) and Pseudomonas aeruginosa (P. aeruginosa) (27.69%) were the most identified microorganisms in VAP cases. SOFA (p-value < 0.0001) and APACHE II (p-value < 0.0001) scores were effective in assessing the severity of illness and predicting mortality in VAP patients. Additionally, our investigation highlighted the prognostic potential of CRP levels (odds ratio [OR]: 0.980, 95% confidence interval [CI] 0.968 to 0.992, p = 0.001). Elevated levels of CRP were associated with reduced survival probabilities in VAP patients. Conclusion: This study highlights the microbial profile of VAP and the importance of biomarkers and illness severity scores in predicting survival. Conclusions: The findings emphasize the need for appropriate management strategies to combat microbial resistance and improve outcomes in VAP patients. Full article

The premortem understanding of the role of feline coronavirus (FeCoV) in the lungs of cats is limited as viruses are seldom inspected in the bronchoalveolar lavage (BAL) specimens of small animal patients. This study retrospectively analyzed the prevalence of FeCoV in BAL samples from cats with atypical lower airway and lung disease, as well as the clinical characteristics, diagnostic findings, and follow-up information. Of 1162 clinical samples submitted for FeCoV RT-nPCR, 25 were BAL fluid. After excluding 1 case with chronic aspiration, FeCoV was found in 3/24 (13%) BAL specimens, with 2 having immunofluorescence staining confirming the presence of FeCoV within the cytoplasm of alveolar macrophages. The cats with FeCoV in BAL fluid more often had pulmonary nodular lesions (66% vs. 19%, p = 0.14) and multinucleated cells on cytology (100% vs. 48%, p = 0.22) compared to the cats without, but these differences did not reach statistical significance due to the small sample size. Three cats showed an initial positive response to the corticosteroid treatment based on the clinical signs and radiological findings, but the long-term prognosis varied. The clinical suspicion of FeCoV-associated pneumonia or pneumonitis was raised since no other pathogens were found after extensive investigations. Further studies are warranted to investigate the interaction between FeCoV and lung responses in cats. Full article

Introduction: The rapidly contagious process of respiratory viruses such as SARS-CoV-2 makes it urgent to multiply testing for diagnostics to identify the active viral shedding cases (current infection, carriage state or, residual viral RNA) and decrease the risk of transmission to other patients and healthcare professionals. Although nasopharyngeal swabs (NPSs) are the most common specimen type used for COVID-19 diagnosis, they require supervision by a professional, and concerns have been raised regarding healthcare personnel exposure, difficulty in collection, and patient discomfort. Viral RNA can also be detected in specimens such as saliva, blood, bronchoalveolar lavage fluid, sputum, faeces, and urine. This study aimed to provide updated information about the most suitable biological material to diagnose SARS-CoV-2, considering the risk assessment, specialization needed, test cost, complexity of the collection, and sample treatment associated with the different types of specimens. Methods: An extensive search of scientific review articles was made to collect information about the biological specimens to identify SARS-CoV-2 in the urine, sputum, nasopharyngeal, oropharyngeal, bronchoalveolar (BAL), saliva, faeces, and blood. For this purpose, an index score was developed based on seven categories: Materials and Equipment; Infection Risk for the Health Professional; Infection Risk for the Patient; Collection; Cost; Specialized HR; and RNA Extraction Type. Results and Discussion: Each criterion from the index score was quoted from 1.0 to 5.0, and a sum was made to classify which specimen is the best choice to diagnose SARS-CoV-2, according to the chosen parameters. Data indicated that urine specimens are the most elementary biological sample to access. Regarding RNA extraction, NPSs, OPS, and BAL presented the maximum score. However, BAL has the lowest score regarding associated costs. Concerning sputum and saliva, all the aspects were evaluated with a score of 5.0 except for the RNA Extraction Type in sputum. Regarding the total scores of the multiple specimens, the lowest corresponds to BAL with a score of 1.7, followed by blood with 3.1 and NPSs and OPS with 3.6 and 3.7, respectively. Urine and faeces have the same value, 4.4, sputum has 4.9, and the highest and maximum possible value corresponds to saliva with 5.0, making this last specimen the most suitable for all considered parameters. Conclusion: Although OPS and NPSs are the most used specimens, there are better alternatives. Among all the specimens of the respiratory system, saliva is the most cost-effective specimen for performing SARS-CoV-2 diagnosis. Even though these infections are usually diagnosed clinically based on symptoms and local epidemiology, the identification of the specific pathogen may affect clinical management and be crucial for containing potential outbreaks. Full article

(1) Background: The number of horses suffering from chronic respiratory diseases, resembling human asthma, is increasing but there is still a lack of reliable and accurate methods to detect these disorders. Numerous studies have found elevated plasma concentrations of one of the myokines, namely, myostatin (MSTN), in people suffering from severe asthma. MSTN normally inhibits myoblast proliferation and differentiation through autocrine or paracrine signals. Therefore, given the pathogenesis of asthma, we hypothesize that MSTN could be a useful biomarker of equine asthma. Thus, this study aimed to compare the concentration of MSTN in the blood plasma of fully healthy and asthmatic horses. (2) Methods: A total of 61 horses were clinically examined to confirm or exclude the occurrence of equine asthma, including bronchoalveolar lavage (BAL) fluid cytology performed on 49 horses. This study included three groups of horses, two of which were clinically healthy, and one of which was asthmatic. (3) Results: The mean circulatory MSTN concentration determined using the ELISA method in asthmatic horses was significantly higher than that in clinically healthy young Thoroughbred racehorses (p < 0.05), but it did not differ as compared to the group of healthy, adult leisure horses. (4) Conclusions: The obtained results did not unambiguously support our original hypothesis that MSTM may be a reliable marker for the early diagnosis of equine asthma. To the best of the authors’ knowledge, this is the first study to analyze the plasma MSTN concentration in equine asthma patients, and therefore further studies are needed to confirm our novel findings. Full article

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein–peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy. Full article