Non-invasive detection of Aspergillosis in ventilated patients: Galactomannan analysis in exhaled breath.

This study evaluates the non-invasive diagnosis of Invasive Aspergillosis Pneumonia (IPA) in mechanically ventilated patients by measuring galactomannan (GM) in exhaled breath condensate (EBC). Utilizing a rat model and a novel EBC collection device, we compared GM levels in bronchoalveolar lavage fluid (BALF) and EBC, supplemented by cytokine profiling. Analysis of 75 patients confirmed the device's efficacy, with EBC-GM and BALF-GM showing high diagnostic accuracy (AUC = 0.88). The threshold of 0.235 ng/ml for EBC-GM achieved 92.8 % sensitivity and 66.7 % specificity, with a strong correlation (r = 0.707, P < 0.001) with BALF-GM. This approach offers a safe, effective alternative to invasive diagnostics, enhancing precision with IL-6 and TNF-α measurements. The number registered on clinicaltrails.gov is NCT06333379.

Organizing Pneumonia With Diffuse Alveolar Hemorrhage Induced by the Kampo Medicine Choreito.

Kampo medicine, a traditional Japanese herbal medicine, is covered by the Japanese National Health Insurance and prescribed for various purposes. While relatively safe with few adverse effects, it may potentially cause severe adverse effects, such as lung injury. Herein, we describe the case of a 61-year-old Japanese woman with choreito-induced lung injury that manifested as organizing pneumonia (OP) with diffuse alveolar hemorrhage (DAH). She was referred to our department due to multiple abnormal opacities detected on annual chest radiography. Chest computed tomography (CT) revealed multiple nodules in bilateral lungs. Bloody bronchoalveolar lavage fluid was obtained from the left lingular lobe, appearing nearly normal, while a transbronchial lung biopsy from a subpleural nodule in the left lower lobe was pathologically consistent with OP. The drug lymphocyte stimulation test result was positive for choreito, which the patient had regularly consumed for 6 - 7 months to treat hematuria. Consequently, a diagnosis of choreito-induced OP and DAH was made. Owing to the discontinuation of choreito alone and without the introduction of systemic steroid therapy, the multiple nodules shrank and eventually disappeared on follow-up chest CT. Regardless of the type of crude drug used in Kampo medicine, clinicians must always be careful for potential lung injury, which may present as OP with DAH.

Neutrophilia in the bronchoalveolar lavage fluid increases coughing during flexible fiberoptic bronchoscopy in a pediatric cohort.

Objective: This study is an addition to the already published prospective randomized double-blinded trial by Tschiedel et al. that compared two different sedation regimes in fiberoptic flexible bronchoscopy in pediatric subjects. The objective of the presented study is to analyze the correlation between the neutrophil percentage of the bronchoalveolar lavage fluid (BALF) and coughing episodes during bronchoscopy. Methods: Fifty subjects, aged 1-17 years, received flexible fiberoptic bronchoscopy under deep sedation. The BALF of 39 subjects was analyzed with reference to cytology and microbiology. Results: The percentage of neutrophils from the total cell count ranged from 0% to 95.3% (median 2.7). Nineteen patients (49%) had a percentage of ≥3.0%. Pearson's correlation showed a high correlation (r = 0.529, p = 0.001) between the coughing episodes per minute and the neutrophil percentage in the BALF. Analysis of variance showed a significant difference in neutrophil percentage between the indication groups (p = 0.013). The t-test (p = 0.019) showed a significant difference between the neutrophil percentage for patients with a probable airway infection under immunosuppression (median 2.9) and patients with cystic fibrosis (median 49.6). The linear regression analysis showed a significantly stronger impact of the neutrophil percentage on coughing frequency than the sedation regime (ßneutrophils = 0.526 with p = 0.001 vs. ßsedation = 0.165 with p = 0.251). Conclusion: When bronchoscopy is to be performed on a pediatric patient with suspected bacterial or viral infection, and therefore neutrophilic airway inflammation, coughing is to be expected.

Navigating Diagnostic Pitfalls: False Positivity in GeneXpert Mycobacterium Tuberculosis/Rifampicin Assay.

Tuberculosis (TB), which is predominantly caused by Mycobacterium tuberculosis (MTB), poses severe diagnostic hurdles, especially with pulmonary tuberculosis (PTB), which spreads by aerosols. Sputum culture, the gold standard for MTB diagnosis, is time-consuming, expensive, and easily contaminated. The GeneXpert MTB/RIF (Xpert) assay, a molecular diagnostic tool, can quickly detect MTB and rifampicin (RIF) resistance. However, the ability to identify both live and non-viable MTB DNA, for example, in patients with a previous history of pulmonary tuberculosis or sampling from a contaminated bronchoscope, can result in false positives, as demonstrated in this case series. We present three cases of PTB diagnosed with Xpert, each with no conventional TB symptoms.

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.

Diagnostic performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in bronchoalveolar lavage fluid for pulmonary tuberculosis in HIV-infected patients.

Introduction: Pulmonary tuberculosis (PTB) remains a significant health concern, particularly in individuals infected with human immunodeficiency virus (HIV) who are more susceptible to developing active TB disease. Early and accurate diagnosis of TB is crucial for effective treatment and prevention of transmission. This study aims to evaluate the potential of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of bronchoalveolar lavage fluid (BALF) for diagnosis of suspected PTB in HIV-infected patients. Methods: This retrospective study recruited 60 HIV-infected patients with suspected PTB presenting with respiratory symptoms and abnormal chest radiographs between January 2022 and June 2023. BALF samples were collected and subjected to analysis using MALDI-TOF MS, GeneXpert, acid-fast bacilli (AFB) smear and culture. And their diagnostic performance was compared. Results: The sensitivity of MALDI⁃TOFMS for diagnosing PTB was 83.3 %, which was better than that of smear 11.9 %, culture 40.5 % or Xpert38.1 % (all p < 0.01). The area under the curve (AUC) value of MALDI⁃TOFMS was 0.889, which was better than that of smear 0.532, culture 0.675 or Xpert 0.690 (all p < 0.01). The katG315 and rpoB-RRDR 511 mutations were detected by the MALDI⁃TOFMS in two patients. Conclusion: Nucleotide MALDI-TOFMS has a good clinical performance for rapid diagnosis of PTB from BALF samples in HIV infected patients, and detects mutations of TB simultaneously.

A Bibliometric Analysis of the Role and Research Trending of Bronchoalveolar Lavage in the Diagnosis and Treatment of Ventilator-Associated Pneumonia.

Ventilator-associated pneumonia (VAP) is one of the most common complications in intensive care units (ICUs) and negatively affects patient outcomes. Despite its widespread use as a diagnostic and therapeutic measure, the application and effectiveness of bronchoalveolar lavage (BAL) in the management of VAP require further exploration. This study aimed to evaluate the research dynamics, major trends, and scientific networks of BAL in the diagnosis and treatment of VAP using bibliometric analysis. Literature from the Web of Science database on BAL for the diagnosis and treatment of VAP from 1990 to 2024 was screened and analyzed. Keyword co-occurrence, trend analysis, and citation burst analyses were conducted using CiteSpace to identify research hotspots, core authors, institutions, and countries, as well as the evolution of research domains. The bibliometric analysis included 968 publications. Trend analysis indicated growing interest in BAL techniques, particularly in the categories of RESPIRATORY SYSTEM (burst score: 27.82) and MEDICINE, RESEARCH, and EXPERIMENTAL (burst score: 7.41). The co-citation analysis highlighted influential authors in the field, such as Torres (burst score: 9.35), Croce (burst score: 5.86), and Meduri (burst score: 5.71). Keyword analysis results revealed core clusters in the treatment of VAP with BAL, including "nonbronchoscopic lavage" (silhouette value: 0.703), "ICU-acquired infection" (silhouette value: 0.7), and "ventilator-associated tracheobronchitis" (silhouette value: 0.637). Additionally, geographic analysis showed that North America and Europe dominated the research in this field. Recently, research trends regarding protected specimen brushes and quantitative culture techniques have emerged. This study found broad applications of BAL in VAP management, especially in improving diagnostic accuracy and treatment outcomes. Optimized strategies such as improvement of lavage techniques and multidisciplinary collaboration may emerge as potential research hotspots in the future.

A Comprehensive Outlook on Pulmonary Alveolar Proteinosis-A Review.

Pulmonary alveolar proteinosis (PAP) is an ultra-rare disease caused by impaired pulmonary surfactant clearance due to the dysfunction of alveolar macrophages or their signaling pathways. PAP is categorized into autoimmune, congenital, and secondary PAP, with autoimmune PAP being the most prevalent. This article aims to present a comprehensive review of PAP classification, pathogenesis, clinical presentation, diagnostics, and treatment. The literature search was conducted using the PubMed database and a total of 67 articles were selected. The PAP diagnosis is usually based on clinical symptoms, radiological imaging, and bronchoalveolar lavage, with additional GM-CSF antibody tests. The gold standard for PAP treatment is whole-lung lavage. This review presents a summary of the most recent findings concerning pulmonary alveolar proteinosis, pointing out specific features that require further investigation.

Impact of modulators of nitric oxide synthesis on the level of cytokines in bronchoalveolar lavage in experimental antiphospholipid syndrome.

OBJECTIVE: Aim: The aim of the research is to study the cytokine prof i le (IL-1ß, IL 6, TNF-α, IL-4, IL-10) in bronchoalveolar lavage of lungs in experimental APS and its correction with L-arginine and aminoguanidine. PATIENTS AND METHODS: Materials and Methods: Antiphospholipid syndrome was modeled on white female BALB/c mice. L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for its correction. The concentration of cytokines in bronchoalveolar lavage from the lungs was assessed using the ELISA test. RESULTS: Results: It was established that in cases of APS the concentration of proinf l ammatory cytokines IL-1ß, IL-6 and TNF-a increased in 1.9, 2.3 and 6.6 times, respectively, compare to the control. At the same time a decrease of the IL-4 in 1.7 and IL-10 in 1.8 times was found in the APS group compare to the control. L-arginine reduced the level of proinf l ammatory cytokines IL-1ß by 22%, IL-6 - by 36%, and TNF-α - by 23% compare to the animals with APS. At the same time, the level of anti-inf l ammatory cytokines increased: IL-4 - by 46%, IL-10 - by 57% compare to the APS animal group. Aminoguanidine, a selective iNOS inhibitor, did not cause any signif i cant decrease in pro-inf l ammatory cytokines but the level of anti-inf l ammatory cytokines IL-4 increased by 44% and IL-10 - by 49%. CONCLUSION: Conclusions: The precursor of the NO synthesis L-arginine leads to a decrease in the concentrations of IL-1ß, IL-6, TNF-a and an increase of IL-4 and IL-10 compare to the group of BALB/c mice with APS.

Altered Extracellular Vesicle-Derived Protein and microRNA Signatures in Bronchoalveolar Lavage Fluid from Patients with Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease for which there is no cure. Accumulating research results suggest a role for extracellular vesicles (EVs) in the pathogenesis of COPD. This study aimed to uncover the involvement of EVs and their molecular cargo in the progression of COPD by identification of EV-associated protein and microRNA (miRNA) profiles. We isolated EVs from the bronchial alveolar lavage fluid (BALF) of 18 patients with COPD and 11 healthy controls using size-exclusion chromatography. EV isolates were characterized using nanoparticle tracking analysis and protein content. Proteomic analysis revealed a higher abundance of 284 proteins (log2FC > 1) and a lower abundance of 3 proteins (log2FC < -1) in EVs derived from patients with COPD. Ingenuity pathway analysis showed that proteins enriched in COPD-associated EVs trigger inflammatory responses, including neutrophil degranulation. Variances in surface receptors and ligands associated with COPD EVs suggest a preferential interaction with alveolar cells. Small RNAseq analysis identified a higher abundance of ten miRNAs and a lower abundance of one miRNA in EVs from COPD versus controls (Basemean > 100, FDR < 0.05). Our data indicate that the molecular composition of EVs in the BALF of patients with COPD is altered compared to healthy control EVs. Several components in COPD EVs were identified that may perpetuate inflammation and alveolar tissue destruction.

Bronchoalveolar Lavage Fluid Microbiota is Associated with the Diagnosis and Prognosis Evaluation of Lung Cancer.

The gut microbiota and cancer have been demonstrated to be closely related. However, few studies have explored the bronchoalveolar lavage fluid (BALF) microbiota in patients with lung cancer (LC), specifically the microbiota related to progression-free survival (PFS) in LC. A total of 216 BALF samples were collected including 166 LC and 50 benign pulmonary disease (N-LC) samples, and further sequenced using 16S rRNA amplicon sequencing. Enrolled LC patients were followed up, the therapeutic efficacy was assessed, and PFS was calculated. The associated clinical and microbiota sequencing data were deeply analysed. Distinct differences in the microbial profiles were evident in the lower airways of patients with LC and N-LC, which was also found between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). A combined random forest model was built to distinguish NSCLC from SCLC and reached area under curves (AUCs) of 0.919 (95% CI 86.69-97.1%) and 0.893 (95% CI 79.39-99.29%) in the training and test groups, respectively. The lower alpha diversity of the BALF microbiota in NSCLC patients was significantly associated with reduced PFS, although this link was not observed in SCLC. Specifically, NSCLC with a higher abundance of f_Lachnospiraceae, s_Prevotella nigrescens and f_[Mogibacteriaceae] achieved longer PFS. The enrichment of o_Streptophyta and g_Prevotella was observed in SCLC with worse PFS. This study provided a detailed description of the characteristics of BALF microbiota in patients with NSCLC and SCLC simultaneously and provided insights into the role of the diagnosis and prognosis evaluation. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00135-9.

Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.

This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Mycoplasma species, Pasteurella multocida, and Truepurella pyogenes). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of Mannheimia haemolytica in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.

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.

Diagnostic yield of flexible bronchoscopy for immunocompromised patients with lung infiltrates: A single-center, retrospective study.

BACKGROUND: Pulmonary complications are associated with mortality in immunocompromised patients. The usefulness of bronchoscopy has been reported. However, clinical factors and procedures that influence diagnostic yield are still not established. MATERIALS AND METHODS: We retrospectively analyzed 115 bronchoscopies performed on 108 immunocompromised patients, defined as those who take corticosteroids and/or immunosuppressants. We evaluated clinical factors, sampling procedures, final diagnosis, and severe complications of bronchoscopy. RESULTS: The clinical diagnosis was obtained in 51 patients (44%). Of those, 33 cases were diagnosed as infectious diseases and 18 as non-infectious diseases. Nine out of 115 cases (7.8%) initiated new immunosuppressive treatment for an underlying disorder based on the negative microbiological results obtained with bronchoscopy. Collagen vascular disease was the most common underlying disorders (62 patients, 54%). Bronchoscopy was useful regardless of whether the patient was immunosuppressed to treat collagen vascular disease (P = 0.47). Performing transbronchial biopsy correlated with better diagnostic yield of bronchoscopy (54.7% vs 35.5%, P = 0.049). Other clinical factors, such as radiological findings, respiratory failure or antibiotic use at the time of bronchoscopy did not significantly influence diagnostic yield. Respiratory failure requiring intubation after bronchoscopy occurred only in one case (0.9%). CONCLUSIONS: Our study implied the transbronchial biopsy may be a useful procedure for reaching a diagnosis in immunocompromised patients with pulmonary infiltrates. In addition, our data suggest the usefulness of bronchoscopy for immunocompromised patients due to the treatment of collagen vascular disease as well as other underlying disorders.

Metagenomic next generation sequencing of bronchoalveolar lavage fluids for the identification of pathogens in patients with pulmonary infection: A retrospective study.

Due to the limitations of traditional laboratory methods (TMs), identification of causative pathogens of numerous pulmonary infections (PIs) remains difficult. This study evaluated the value of metagenomic next generation sequencing (mNGS) in the identification of various respiratory pathogens. A total of 207 patients with TMs and mNGS data were collected for this retrospective study. TMs included sputum culture, blood, and bronchoalveolar lavage fluid (BALF) analysis, or polymerase chain reaction analysis of throat swabs. Otherwise, BALF was collected and analyzed using mNGS. For bacterial pathogens, sensitivities of mNGS as compared to TMs were 76.74 % and 58.14 % (P=0.012). For fungal pathogens, the detection rate of mNGS sensitivity was higher as compared to that of TMs (93.68 % vs 22.11 %; P<0.001). The positive predictive value and negative predictive value were also greater for mNGS. Use of mNGS for BALF analysis offers good specificity and thus facilitates to the clinical diagnosis of PIs.

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.

An integrated metaproteomics workflow for studying host-microbe dynamics in bronchoalveolar lavage samples applied to cystic fibrosis disease.

Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our understanding of host-microbe interactions, we have developed an integrated analytical and bioinformatic mass spectrometry (MS)-based metaproteomics workflow to analyze clinical bronchoalveolar lavage (BAL) samples from people with airway disease. Proteins from BAL cellular pellets were processed and pooled together in groups categorized by disease status (CF vs. non-CF) and bacterial diversity, based on previously performed small subunit rRNA sequencing data. Proteins from each pooled sample group were digested and subjected to liquid chromatography tandem mass spectrometry (MS/MS). MS/MS spectra were matched to human and bacterial peptide sequences leveraging a bioinformatic workflow using a metagenomics-guided protein sequence database and rigorous evaluation. Label-free quantification revealed differentially abundant human peptides from proteins with known roles in CF, like neutrophil elastase and collagenase, and proteins with lesser-known roles in CF, including apolipoproteins. Differentially abundant bacterial peptides were identified from known CF pathogens (e.g., Pseudomonas), as well as other taxa with potentially novel roles in CF. We used this host-microbe peptide panel for targeted parallel-reaction monitoring validation, demonstrating for the first time an MS-based assay effective for quantifying host-microbe protein dynamics within BAL cells from individual CF patients. Our integrated bioinformatic and analytical workflow combining discovery, verification, and validation should prove useful for diverse studies to characterize microbial contributors in airway diseases. Furthermore, we describe a promising preliminary panel of differentially abundant microbe and host peptide sequences for further study as potential markers of host-microbe relationships in CF disease pathogenesis.IMPORTANCEIdentifying microbial pathogenic contributors and dysregulated human responses in airway disease, such as CF, is critical to understanding disease progression and developing more effective treatments. To this end, characterizing the proteins expressed from bacterial microbes and human host cells during disease progression can provide valuable new insights. We describe here a new method to confidently detect and monitor abundance changes of both microbe and host proteins from challenging BAL samples commonly collected from CF patients. Our method uses both state-of-the art mass spectrometry-based instrumentation to detect proteins present in these samples and customized bioinformatic software tools to analyze the data and characterize detected proteins and their association with CF. We demonstrate the use of this method to characterize microbe and host proteins from individual BAL samples, paving the way for a new approach to understand molecular contributors to CF and other diseases of the airway.

Feasibility of the modified mini-bronchoalveolar lavage techniques using a nasogastric suction catheter and polytetrafluoroethylene bronchoscopic suction catheter in diagnosing bilateral pneumonia: a pilot study.

Background: The adequacy of actual lower respiratory tract samples collected using the current collection technique is debated. Endotracheal aspiration is commonly insufficient and can be contaminated with colonization from the proximal airway. Diagnostic bronchoscopy is the standard method for collecting specimens from the lower respiratory tract. However, it is usually unavailable in resource-limited settings. At present, noninvasive methods with the mini-bronchoalveolar lavage (BAL) catheter are used to collect specimens from the lower respiratory tract. Compared with the nasogastric (NG) tube, the polytetrafluoroethylene (PTFE) catheter, a modified mini-BAL catheter that suctions the more distal part of the tracheobronchial tree, can collect actual lower respiratory tract specimens. Methods: This prospective open-label pilot study included patients aged >18 years who were diagnosed with bilateral pneumonia and who required mechanical ventilation. Lower respiratory tract samples were collected via endotracheal aspiration, mini-BAL using an NG tube, and mini-BAL using a PTFE bronchoscopic catheter. Data on return fluid volume, white blood cell (WBC) count, microbiologic information obtained via quantitative culture, and each procedure-related complication were recorded. Results: The return fluid volumes of the NG tube and PTFE groups were 50 and 40 mL, respectively. The median WBC counts were 245 cells/cumm3 in the NG tube group and 305 cells/cumm3 in the PTFE group. Culture from endotracheal aspiration detected polymicrobial organisms in 8 (20.0%) patients. Further, 19 (47.5%) patients in the NG tube group and 18 (45.0%) in the PTFE group presented with polymicrobial organisms. Approximately 10% of patients developed mini-BAL-related complications, including arrhythmia (2.5%), mild hypoxemia (2.5%), and mild bleeding (5.0%). Conclusions: The two modified mini-BAL techniques are feasible in diagnosing patients with pneumonia requiring mechanical ventilation. The mini-BAL technique is more likely to detect polymicrobial organisms compared with endotracheal aspiration, which can then identify the causative polymicrobial organism of ventilator associated pneumonia (VAP) and lead to antibiotic adjustment. Moreover, it is easy to perform, can yield adequate specimens, and has few complications.

Exploring a Rare Pulmonary Coinfection: Cryptococcal Pneumonia and Exophiala dermatitidis in an Immunocompetent Host.

Pulmonary cryptococcosis is becoming increasingly common in immunocompetent hosts, manifesting with variable clinical presentations ranging from asymptomatic colonization to severe pneumonia. Radiological findings are non-specific, such as nodular infiltrates, mass-like lesions, and mediastinal lymphadenopathy. We present a case of a 61-year-old woman with Cryptococcus neoformans pneumonia coinfected with Exophiala dermatitidis, an unusual occurrence in an immunocompetent host and the first of its kind. This coinfection posed significant diagnostic challenges due to the rare occurrence of each individual organism in immunocompetent patients as well as the difficulty of their laboratory diagnosis. Treatment regimens, particularly in coinfections, warrant careful consideration to mitigate mortality risk. This case underscores the importance of comprehensive diagnostic strategies and optimized treatment regimens for rare fungal coinfections in immunocompetent hosts.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.