BAL Samples Research Articles

Diagnosis of pulmonary lophomoniasis infection in patient with systemic lupus erythematosus; A case report and literature review.

Over the past 30 years, there has been an increasing number of documented human infections associated with the protozoan Lophomonas, specifically Lophomonas blattarum, which is considered a relatively rare infection. These infections are primarily associated with states of immune suppression, including those resulting from corticosteroid therapy. We report a 61-year-old female patient with a 20-year medical history of Systemic lupus erythematosus (SLE) who was admitted due to persistent respiratory symptoms that were unresponsive to treatment. The patient was receiving immunosuppressive therapy for SLE. Upon hospitalization, computed tomography of the lungs revealed the presence of centrilobular nodules exhibiting tree-in-bud patterns, as well as bronchiectasis, predominantly in the middle and lower lobes. Subsequently, the patient underwent bronchoscopy, during which a BAL sample was obtained. Microscopic analysis of the sample indicated the presence of L. blattarum. Clinicians often focus on the primary symptoms of SLE, but they must also consider the risk of severe respiratory complications like lophomoniasis. This condition is critical to address in the management of SLE patients, who are immunosuppressed due to the disease's nature and its treatment.

Intracranial dissemination of Klebsiella pneumoniae originating from pulmonary infection: a case report

BackgroundMetastatic brain abscesses caused by Klebsiella pneumoniae are extremely rare but life-threatening conditions. To depict a unique case of the middle-aged hypertensive man with an unusual presentation of metastatic brain abscesses originating from a pleural abscess caused by Klebsiella pneumoniae and subsequently leading to loss of consciousness (LOC).Case reportA 52-year-old Iranian man with a history of hypertension presented to the emergency department with a five-day history of worsening cough, high-grade fever, shortness of breath, chest pain, fatigue, and a productive cough. Laboratory tests revealed leukocytosis, elevated C-reactive protein, and respiratory alkalosis. A chest computed tomography scan confirmed pneumonia, and a brain scan revealed multiple hypodense lesions. Despite antibiotic therapy, the patient's condition worsened, leading to confusion, disorientation, and loss of consciousness. Magnetic resonance imaging revealed multiple ring-enhancing lesions, suggesting an abscess formation. Bronchial washings and BAL samples confirmed a lower respiratory tract infection. Cultures from the bronchial washings grew Klebsiella pneumoniae.ConclusionsMetastatic brain abscesses caused by Klebsiella pneumoniae are exceedingly rare but life-threatening conditions. Timely diagnosis and effective antimicrobial treatment are critical for patient outcomes. This case underscores the significance of recognizing atypical presentations of bacterial infections, as early detection and appropriate management can significantly impact patient outcomes.

Skin Rash with Acute Respiratory Distress Syndrome in Chronic Miliary Tuberculosis: The Role of HRCT Thorax and Bronchoscopy as Diagnostic Tools

Pulmonary tuberculosis (TB) is a leading cause of mortality and morbidity in India, presenting with diverse clinical and radiological manifestations such as consolidation, cavitation, lymphadenopathy, pleural effusion, miliary TB, and endobronchial TB. While acute miliary TB presenting as Acute respiratory distress syndrome (ARDS) is documented, the association with a skin rash mimicking viral exanthematous fever is not. This case report describes the case of a 41-year-old male patient with acute dyspnea, a maculopapular rash, and lung infiltrates on chest radiograph, initially diagnosed with acute hypoxic respiratory failure and later confirmed as ARDS. High-resolution computed tomography (HRCT) thorax revealed consolidation with nodules and a characteristic butterfly pattern sparing the apices and bases. Bronchoscopy confirmed TB etiology through MTB genome in BAL samples. The patient improved significantly with intravenous antibiotics, steroids, and HFNC, leading to a documented cure after 6 months of anti-tuberculosis treatment. This report highlights the importance of considering TB in cases with nodular consolidation and positive limiting signs and recommends ICU bronchoscopy as a reliable diagnostic tool. The Kigali modification for ARDS definition was instrumental in managing this case. Use of Kigali modification for defining ARDS in resource limited setting guided management in our case.

Changes in urinary glutathione sulfonamide (GSA) levels between admission and discharge of patients with cystic fibrosis

There is an urgent need to develop sensitive, non-invasive biomarkers that can track airway inflammatory activity for patients with cystic fibrosis (CF). Urinary glutathione sulfonamide (GSA) levels correlate well with GSA levels in BAL samples and other markers of neutrophilic inflammation, suggesting that this biomarker may be suitable for tracking disease activity in this population. We recruited 102 children (median 11.5 years-old) and 64 adults (median 32.5 years-old) who were admitted to hospital for management of an acute pulmonary exacerbation and/or eradication of infectious agents such as Pseudomonas aeruginosa or Staphylococcus aureus. Our aim was to explore how urinary GSA levels changed across admission timepoints. Urine samples were collected at admission and discharge, and GSA measured by liquid chromatography with mass spectrometry. Paired admission-discharge results were compared using Wilcoxon signed-rank test. Paired admission-discharge samples were available for 53 children and 60 adults. A statistically significant difference was observed between admission-discharge for children and adults. Spearman's correlation analysis identified a correlation between urinary GSA levels and sex and S. aureus infection for children only. Our preliminary findings suggest that urinary GSA is responsive to the resolution of an acute pulmonary exacerbation and therefore warrants further studies in this population.

Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed.

Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh-EVs). Using our customizable antibody-purification assay, EV-CATCHER, we initially determined that exh-EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh-EVs harbour lung-specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh-EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV-CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti-CCSP/SFTPC EV-CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh-EV subpopulations from EBC allows non-invasive sampling of EVs produced by lung tissues.

Role of BAL and Serum Krebs von den Lungen-6 (KL-6) in Patients with Pulmonary Fibrosis.

Background: Interstitial lung diseases (ILDs) encompass a diverse group of disorders affecting the lung interstitium, leading to inflammation, fibrosis, and impaired respiratory function. Currently, the identification of new diagnostic and prognostic biomarkers for ILDs turns out to be necessary. Several studies show the role of KL-6 in various types of interstitial lung disease and suggest that serum KL-6 levels can be used as a prognostic marker of disease. The aim of this study was to analyze KL-6 expression either in serum or bronchoalveolar lavage samples in order to: (i) make a serum vs. BAL comparison; (ii) better understand the local behavior of fibrosis vs. the systemic one; and (iii) evaluate any differences in patients with progressive fibrosis (PPF) versus patients with non-progressive fibrosis (nPPF). Methods: We used qRT-PCR to detect KL-6 expression both in serum and BAL samples. Mann-Whitney's U test was used to compare the differential expression between groups. Results: In serum, KL-6 is more highly expressed in PPF than in non-progressive fibrosis (p = 0.0295). This difference is even more significant in BAL (p < 0.001). Therefore, it is clear that KL-6 values are related to disease progression. Significant differences were found by making a comparison between BAL and serum. KL-6 was markedly higher in serum than BAL (p = 0.0146). Conclusions: This study identifies KL-6 as a promising biomarker for the severity of the fibrosing process and disease progression in ILDs, with significantly higher levels observed in PPF compared to nPPF. Moreover, the marked difference in KL-6 levels between serum and BAL emphasizes its potential diagnostic and prognostic relevance, providing enlightening insights into both the local and systemic aspects of ILDs.

ОСОБЕННОСТИ ТЕЧЕНИЯ МИКОБАКТЕРИОЗА ЛЕГКИХ В ЗАВИСИМОСТИ ОТ ВЫЯВЛЕНИЯ ВОЗБУДИТЕЛЯ В МОКРОТЕ

Objective: to evaluate treatment effectiveness in patients with mycobacterial pulmonary disease and its association with mycobacteria detection in sputum. Materials and methods. The study enrolled 196 patients with confirmed mycobacterial pulmonary disease. The diagnosis was established as per ATS/IDSA criteria (2007). The patients were divided into two groups: group 1 consisted of 108 patients with nontuberculous mycobacteria (NTM) detected in sputum; group 2 consisted of 60 patients with NTM detected in BAL and/or lung tissue samples. Female patients prevailed (78.6%). The average patients’ age was 56.9 ± 11.3 years. The time period from diagnosing pulmonary mycobacterial disease to commencementof combination antibacterial therapy varied from one month to 3.5 years. Patients were followed up during one year after treatment completion. Results. The most commonly identified NTM were MAC (M. аvium, M. intracellulare) and M. kansasii. The combination antibacterial therapy was administered to 168 (85.7%) patients. Clinical cure was more frequently achieved in the patients of group 2 compared to group 1 (21.7 and 3.7% respectively, р &lt; 0.01). The frequency of clinical improvement without conversion of biological samples did not significantly differ between the groups (40 and 54.6% respectively, …). The frequency of relapse was similar in both groups and did not exceed 5%. Conclusion. The presence of NTM in BAL while the absence of NTM in sputum allows predicting more favourable disease course and better response to treatment.

Comparison of diagnostic techniques in patients with COVID-19-associated pulmonary aspergillosis (CAPA) in a Venezuelan population

Background: Invasive aspergillosis related to COVID-19 or CAPA is defined by the association of a group of histological, mycological, radiological and clinical criteria that establish this disease as a secondary infection of high morbidity without timely diagnosis. Clinical and histological methods, due to the non-specific characterization of the disease, have been supported by techniques based on microbiological methods, such as culture and direct examination, with a sensitivity close to 50%, allowing the identification of biomarkers such as the GM of the pathogen, being the immunological techniques of greater sensitivity and specificity, the use of radial immunodiffusion and ELISA with antigens secreted by Aspergillus spp. are recommended. The objective of this article was to compare three different serological diagnostic methods used in the mycology laboratory to diagnose CAPA in a Venezuelan population. Methods: Based on a longitudinal study, 77 patient samples diagnosed as positive for SARS-CoV-2 by RT-PCR were analyzed, of which 24 CAPA BAL samples were subsequently evaluated by conventional mycological diagnostic methods (direct examination and culture) and corresponding CAPA serum samples by three serological methods: DID, ELISA with Aspergillus pool antigen, and the Galactomannan detection with Lateral flow (GM- LFA) essay. Results: By microscopy, fungal structures were observed in 18/24 patients and 16/24 were positive for GM-LFA. Regarding the DID assays, precipitating bands were obtained in 21/24 patients with 86% sensitivity; and 97% specificity. To use ELISA technique, shows that 16/24 sera from patients with aspergillosis were positive, which represents 66% sensitivity and 97% specificity. As for GM- LFA, all sera were tested and 16/24 positive sera were obtained with the commercial kit, representing 66% sensitivity and 97% specificity. Conclusions: Finally, when comparing the three tests studied, all techniques were found to be highly sensitive and have excellent specificity, recommending that the diagnosis of CAPA and IPA be based on a combination of diagnostic assays.

Effect of volume of instillate on the diagnostic utility of bronchoalveolar lavage galactomannan in patients with suspected chronic pulmonary aspergillosis-A pilot study.

Bronchoalveolar lavage (BAL) galactomannan (GM) is commonly used to diagnose Aspergillus-related lung diseases. However, unlike serum GM, which is measured in undiluted blood, BAL-GM is estimated using variable aliquots and cumulative volume of instillates during bronchoscopy. Since different studies have reported varying diagnostic accuracy and cut-offs for BAL-GM in CPA, we hypothesized that the total volume of instillate and 'order/label' of aliquots significantly affects the BAL-GM values, which was evaluated as part of this study. We obtained 250 BAL samples from 50 patients (five from each) with suspected chronic pulmonary aspergillosis. BAL fluid was collected after instilling sequential volumes of 40 mL of normal saline each for the first four labels and a fifth label was prepared by mixing 1 mL from each of the previous labels. The GM level of each label was measured by PLATELIA™ ASPERGILLUS Ag enzyme immunoassay. This study measured the discordance, level of agreement, diagnostic characteristics (sensitivity, specificity and AUROC) and best cut-offs for BAL-GM in the different aliquots of lavage fluid. The study population, classified into CPA (28%) and non-CPA (72%) groups, based on ERS/ESCMID criteria (excluding BAL-GM) were not different with respect to clinico-radiological characteristics. The discordance of BAL-GM positivity (using a cut-off of >1) between the serial labels for the same patient ranged between 10% and 22%, while the discordance between classification using BAL-GM positivity (using a cut-off of ≥1) and clinic-radio-microbiological classification ranged between 18% and 30%. The level of agreement for serial labels was at best fair (<0.6 for all except one 'label'). The AUROC for the serial samples ranged between 0.595 and 0.702, with the '40 mL and the 'mix' samples performing the best. The best BAL-GM cut-off also showed significant variation between serial labels of varying dilutions (Range:1.01 - 4.26). This study highlights the variation in BAL-GM measured and the 'positivity' between different 'labels' of aliquots of BAL, with the first aliquot and the mixed sample showing the best performances for diagnosis of CPA. Future studies should attempt to 'standardise' the instilled volume for BAL-GM estimation to standardise the diagnostic yield.

The role of Xpert MTB/RIF using bronchoalveolar lavage fluid in active screening: insights from a tuberculosis outbreak in a junior school in eastern China.

Tuberculosis (TB) outbreaks in schools present a public health challenge. In order to effectively control the spread of transmission, timely screening, accurate diagnosis and comprehensive epidemiological investigations are essential. In July 2021, a TB outbreak occurred in a junior high school in Y City, Zhejiang Province. Students and faculty were screened for TB by symptom screening, chest radiography, and tuberculin skin test during four rounds of contact screenings. For sputum smear-negative and sputum-scarce patients, bronchoscopy was used to collect BAL samples for Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF). Whole-genome sequencing and bioinformatics analysis were performed on isolates to identify the strains of MTB isolates and predict drug resistance. Between July 2021 and November 2021, a total of 1,257 students and faculty were screened for TB during screenings. A total of 15 students (1.2% of persons screened) aged 15 years were diagnosed with TB. Eighty percent (12/15) of the cases were laboratory-confirmed (10/12 [83%] Xpert MTB/RIF-positive, 2/12 [17%] culture-positive). Most cases (12/15 [80%]) were in students from Class 2. All cases were asymptomatic except for the index case who had symptoms for more than two months. Seven MTB isolates were collected and belonged to lineage 2. Our findings demonstrated the potential of Xpert MTB/RIF using BAL as a screening tool in school TB outbreaks for sputum smear-negative and sputum-sparse suspects, which may not only rapidly improves diagnostic accuracy, but also facilitates epidemiological investigations and homology analysis.

Lymphopenia and high Ki-67 expression in peripheral blood CD4+ and CD8+ T cells associate with progressive sarcoidosis

BackgroundEarly identification of patients at risk for progressive sarcoidosis may improve intervention. High bronchoalveolar lavage fluid (BALF) lymphocytes and peripheral blood (PB) lymphopenia are associated with worse prognosis. The mechanisms...

621. Evaluation of a Multiplex Endpoint PCR for the Detection of 21 Fungi Commonly Causing Fungal Pneumonia Using Barcoded Magnetic Bead Technology

Abstract Background Fungal pneumonia caused by Aspergillus, Mucorales, and other fungi are potentially life-threatening and often are difficult to diagnose due to similar clinical presentations and poor sensitivity by culture. The BioCode MDx-3000 from Applied BioCode uses Barcoded Magnetic Bead (BMB) technology to digitally detect 21 fungi in a multiplex PCR. We evaluated the BioCode assay using clinically simulated samples and retrospective frozen bronchoalveolar lavage samples. Methods For limit of detection (LOD), 10-20 replicates of each organism were run at 1E4-10 spores/mL. Reproducibility was performed by running three replicates of each target (1E3 spores/mL) within one run, three separate times. Specificity was determined by testing for 37 fungal analytes not targeted by the panel. Matrix specific inhibition was also tested by spiking each organism in the following matrices: whole blood, plasma, serum and BAL. 90 retrospective BAL samples from patients with fungal culture results were run to assess clinical performance. Results LOD of 21 fungal organisms ranging from 19.83 – 271.76 spores/ml. Detection rate was 100% (36/36) in all samples mixed with two fungi; 95% (20/21) in all samples mixed with three fungi. No detection or cross-reactivity was observed in samples containing any of the 37 non-targeted fungi. All replicates were detected in Inter- and intra- reproducibility experiments. No difference or inhibition was observed in detection of targeted fungi spiked in different matrices. Testing retrospective BAL samples showed an overall positive concordance of 61.4% and negative concordance of 98.1%. In 23 BALs positive by both calcofluor white stain (hypha elements) and culture, the BioCode assay correctly detected fungal targets in 78% (18/23) of the samples. Additionally, the assay produced positive detection in 25% of BALs that were culture negative. Limit of Detection and Concordance Limit of detection for the 21 fungal targets. Concordance for each target in retrospective BALs compared to culture. N/A indicates not tested. Conclusion The BioCode assay showed an overall good analytical sensitivity and specificity. Analysis of retrospective BAL samples demonstrated the assay showing good concordance with the culture results. Further studies are warranted to determine the assay’s clinical sensitivity and specificity in aid in diagnosis of fungal pneumonia. Disclosures Kristen Haag, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode|Applied BioCode, Inc.: Stocks/Bonds Michael Aye, PhD, Applied BioCode: Stocks/Bonds Martín Cardona, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode. Sean Zhang, PhD, Applied BioCode: Grant/Research Support|IMMY Diagnostics: Grant/Research Support|KARIUS: Advisor/Consultant|Pearl Diagnostics: Grant/Research Support|Scanogen: Grant/Research Support|T2 Biosystems: Advisor/Consultant|Vela Diagnostics: Grant/Research Support

Lophomonas Infection in Patients with Respiratory Diseases in Southeastern Iran Using Wet Mount, Giemsa and Trichrome Staining

Background: In developing countries, pulmonary infections are one of the major causes of death because the incidence and prevalence of pulmonary diseases have increased dramatically. Several species of protozoa can be found in the respiratory system. Pulmonary protozoan infections are increasingly identified in clinical settings. Protozoans within the genus Lophomonas are endo-commensals of the hindgut of arthropods. Recently the trophozoite forms of Lophomonas have been observed in human tissues. Little is known about the occurrence of these protozoa in the Iranian population. Objective: This study was designed to determine the prevalence of Lophomonas in bronchoalveolar lavage specimens from the patients with respiratory diseases referred in Kerman province, southeast of Iran. Methods: A total of 200 patients were selected by simple random sampling. BAL samples were transferred to the Parasitology lab, direct smears were prepared for each specimen and two staining methods, Giemsa and Trichrome were performed for Lophomonas microscopical identification. The data were analyzed using descriptive statistics and Chi-square test. Results: Lophomonas trophozoites were found in 48 (24%) patients, at least in one of the methods. The mean age of the patients was 58.3 years (58.1 in men, 58.4 in women). Out of 200 samples, 45 (22.5%), 30 (15%), and 11(5.5%) were positive by wet mount microscopy, Giemsa, and Trichrome staining, respectively. Conclusion: This study presented the first finding of Lophomonas infection in patients with pulmo-nary symptoms in southeast Iran.

Etiology of acute respiratory infections using multiplex polymerase chain reaction in children admitted to pediatric intensive care unit: A single-centered retrospective observational study from Western India

Background: Acute respiratory infections (ARIs) are an important cause of pediatric mortality–morbidity worldwide, the most common etiology being viral. This study aims to identify causative organisms for ARIs admitted in pediatric intensive care unit (PICU), when multiplex polymerase chain reaction (PCR) testing of respiratory secretions was sent; any seasonal trends detect microbiological correlation when co-infections. Subjects and Methods: This was a retrospective observational study, from July 2021 to December 2022, of children aged 1 month–18 years, whose multiplex PCR tests (nasopharyngeal, endotracheal [ET] secretion or bronchoscopic alveolar lavage [BAL]) were sent when admitted for ARI to tertiary care PICU. Results: In the study period, 372 of 1492 medical PICU admissions were ARI. Multiplex PCR of 81 respiratory secretions was sent, of which 69 (85%) were positive. Multiplex pcr sample positivity : 83% for nasopharyngeal aspirate, 78% for ET secretions, 100% for BAL samples. Forty-one percent of samples detected &gt;1 organism. Respiratory syncytial virus (RSV)-A was the most common virus (18); other organisms included adenovirus (n = 5), influenza (n = 9), parainfluenza (n = 5), rhinovirus: 13, Pneumocystis Jerovecci (PCP): 4, Streptococcus pneumoniae: 17, pertussis: 1, and Haemophilus influenzae B: 9. ARIs were seen throughout the year with peaks in monsoon season and a peak in cases of ARI due to RSV from July to October. Of co-infections with bacteria in ET secretions and BAL samples via multiplex PCR, bacterial culture reports were sterile. Conclusions: Multiplex PCR detected organisms in 85% of ARI patients tested. Most of the ARIs getting admitted to PICU were viral in origin. RSV was the most common virus isolated showing peak from July to October, local monsoon season. With extended viral and bacterial PCR being available, mixed infections/colonization with uncertain significance are being detected.

Pulmonary Aspergillosis in Naïve Non-neutropenic Lung Cancer Patients

Abstract: Pulmonary aspergillosis is the most common mould infection of the lungs. Patients with chronic lung disease or mild immunodeficiency are at risk. Lung cancer represents an oncological challenge. Limited data are available about aspergillosis in newly diagnosed non-neutropenic lung cancer patients. Objectives:: The aim of this work was to evaluate naïve lung cancer patients for the presence of as-pergillosis, identify fungal isolates, and determine antifungal susceptibility. The inhibitory effect of secondary metabolites of fungal isolates on the human fibroblast cell line was also assessed. Results:: Aspergillus isolates were detected in 33 BAL samples out of the 69 patients (47.8%). The commonest isolate was A. niger. There was no significant correlation between total fungal count and age of patients. The antifungal Micafungin had a broad-spectrum and potent fungistatic activity against all fungal species, with A. niger being the most sensitive. MTT assay was performed to evaluate the cytotoxic effect of both A. niger and A. fumigatus on normal lung cells (WI-38) and re-vealed that toxicity was concentration-dependent and A. niger had significantly lower IC 50, indi-cating more toxicity. Conclusion:: In the context of the diagnosis of lung cancer, maintaining vigilance is crucial to diag-nose fungal infection as aspergillosis. Initiation of proper management can help improve patient outcomes.

May noninvasive mechanical ventilation and/ or continuous positive airway pressure increase the bronchoalveolar lavage salvage in patients with pulmonary diseases? Randomized clinical trial - Study protocol

PurposeBronchoalveolar lavage (BAL) procedure is a useful tool in the diagnosis of patients with interstitial lung disease (ILD) and is helpful in clinical research of chronic obstructive pulmonary disease (COPD) patients. Still little is known about predictors of poor BAL salvage. The trial aims to find the most efficient way to improve BAL recovery. Material and methodsOur study is a prospective, multicenter, international, two-arm randomized controlled trial. We aim to obtain BAL samples from a total number of 300 patients: 150 with ILD and 150 with COPD to achieve a statistical power of 80 ​%. Patients with initial BAL salvage <60 ​% will be randomized into the non-invasive ventilation (NIV) or continuous positive airway pressure (CPAP) arm. The NIV and CPAP will be set according to the study protocol. The influence on BAL salvage will be assessed in terms of BAL volume and content. Multivariable analysis of the additional test results to determine predictors for low BAL recovery will be conducted. In a study subgroup of approximately 20 patients per specific disease, a metabolomic assessment of exhaled air condensate will be performed. All procedures will be assessed in terms of the patient's safety. The trial was registered on clinicaltrials.gov (ID# NCT05631132). Interested experienced centers are invited to join the research group by writing to the corresponding author. ConclusionThe results of our prospective study will address the currently unsolved problem of how to increase BAL salvage in patients with pulmonary diseases without increasing the risk of respiratory failure exacerbation.

Protein N-glycosylation in the bronchoalveolar space differs between never-smokers and long-term smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n=5) and long-term smokers (LTS) with (LTS+, n=4) and without COPD (LTS-, n=8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

AEROBIC BACTERIAL PROFILE AND ANTIBIOGRAM OF BRONCHOALVEOLAR LAVAGES IN PULMONARY INFECTIONS AT A TERTIARY CARE HOSPITAL

Objective: Lower respiratory tract infections (LRTIs) are one of the common clinical problems in community and hospital settings and the commonest causes of morbidity and mortality. In pulmonary infections, BAL fluid sample has high sensitivity and reliability in diagnosis. To determine the distribution of bacterial isolates in BAL samples and antibiotic sensitivity patterns in most frequently isolated bacterial pathogens other than Mycobacterium tuberculosis.&#x0D; Methods: The study was conducted on 218 BAL samples received in the microbiology laboratory, Andhra Medical College from various wards of King George Hospital; Visakhapatnam; Andhra Pradesh, over a period of one year (from January 2022 to December 2022). All samples were processed according to standard microbiology protocols. Antimicrobial susceptibility was tested by the Kirby-Bauer disc diffusion method as per the CLSI guidelines 2022.&#x0D; Results: Total 218 BAL samples were studied. Among 218 BAL samples, 144(66%) samples were from male patients and 74(34%) samples from female patients. Out of 218 samples, 119(55%) were culture positive and 99(45%) were culture negative. Among 119 bacterial isolates, most predominant pathogen was Klebsiella pneumoniae 51(42.8%) followed by Pseudomonas aeruginosa 46(38.6%), Escherichia coli 11(9.2%), Enterobacter species 6(5.0%), Acinetobacter species 3(2.5%) and Staphylococcus aureus 2(1.68%). Out of these isolated pathogens, Gram-negative bacilli were more. These gram-negative isolates were most sensitive to Piperacillin-Tazobactam (74.3%) followed by Meropenem (71.7%), Amikacin (60.6%) and least sensitive to Ceftazidime (39.3%) and Cefotaxime (37.6%).&#x0D; Conclusion: The present study shows that gram-negative bacilli were more commonly isolated in BAL samples and the bacterial isolates were most sensitive to Piperacillin-Tazobactam and Meropenem and highly resistance to Cefotaxime and Ceftazidime. Hence, formulating a regular antibiogram in hospitals will help in developing local antibiotic policies, which may provide better patient management and judicious use of antibiotics by clinicians to prevent the risk of the emergence of multidrug-resistant pathogens.

131 Alveolar Macrophage Degranulation Initiates the Spatially Restricted Innate Immune Response During Ventilator-Assisted Nanoparticle Inhalation

Abstract To visualize and measure in real-time the cellular pulmonary innate immune response elicited by inhaled NPs, we apply state-of-the-art intravital microscopy on the alveolar region of the murine lung. Fluorescent carboxyl-quantum-dot-NPs (deposited dose of 16 cm2/g) applied via ventilator-assisted inhalation accumulated within seconds as distinct fluorescent spots at the alveolar walls. Already at 30min, cQD-NPs elicited an increase in microvascular neutrophil numbers as compared to controls, which subsequently transmigrated into the alveolar space. This early immune response was not specific to cQD-NPs, since a comparable increase in neutrophil numbers was also observed upon inhalation of a bioequivalent dose of carbon black NPs, a typical component of urban air pollution. AMs actively accumulated cQD-NPs over time, starting few minutes after inhalation. Significantly more neutrophils were attracted in proximity to cQD-NP-laden AMs, as compared to NP-free AMs or control areas with epithelial associated cQD-NP. Prior airway application of a cellular degranulation inhibitor prevented cQD-NP and CNP induced neutrophil recruitment at 2h and 24 h after inhalation. Inhibition of Fcγ-receptor mediated AM cQD-NP uptake by prior application of anti-CD64 mAbs or neutralizing of TNFα by anti-TNFα mAbs in the airspace, abolished this early immune response. Importantly, initial ImageStream flow cytometry analysis of BAL samples indicated an increase of macrophage-derived microvesicles upon NP inhalation. Taken together, our data links NP-induced AM degranulation and cytokine release to the rapid and site-specific recruitment of neutrophils, suggesting this process to be a key event in mounting the immune response upon NP inhalation.

CASE SERIES OF ISOLATED PULMONARY NOCARDIOSIS AMONG POST-COVID-19 INDIVIDUALS

Nocardia infections are rare in an immunocompetent and healthy individual. It is frequently diagnosed and reported very late during the disease. Here we present an interesting case series of isolated pulmonary nocardiosis among post COVID-19 patients. A 45-year-old male presented to general medicine OPD with breathlessness and cough with expectoration. The patient was a known case of diabetes. A Sputum sample was sent for culture, gram, and acid-fast stain, and the nasopharyngeal swab was sent for RT-PCR and turned out to be COVID-19 negative. Gram stain showed numerous pus cells with filamentous hyphae-like structures. Nocardiosis was strongly considered based on imaging morphology. A 65-year-old female presented to OPD with fever, respiratory distress, and expectorating cough for one month. The patient was a not known case of diabetes and hypertension. The patient was diagnosed with pneumonia and had a history of COVID-19. The microscopic examination of the sputum sample revealed no fungal elements. The Gram and Ziehl-Neelsen stain showed pus cells with filamentous bacilli and no acid-fast bacilli. A 50-year-old male presented to general medicine OPD with breathlessness and expectorating cough for four weeks. The patient was a not known case of diabetes and hypertension. The sputum sample was negative for ZN stain; the Gram stain showed numerous pus cells with filamentous hyphae-like structures. The modified acid-fast staining was done for sputum and BAL samples in all cases showed filamentous weak acid-fast bacilli resembling Nocardia species. The patient did well with cotrimoxazole and was discharged later. The differential diagnosis of nocardia should always be in mind in case of lung infections. All respiratory samples must also be screened for Nocardia. The early diagnosis and management of pulmonary nocardiosis decide the patient’s prognosis.