Lavage Samples Research Articles

Neutrophil-Derived Peptidyl Arginine Deiminase Activity Contributes to Pulmonary Emphysema by Enhancing Elastin Degradation.

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and invitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Periostin Is a Biomarker for Anterior Shoulder Instability: Proteomic Analysis of Synovial Fluid

Background: The incremental biological changes in the synovial microenvironment of the shoulder in acute and chronic instability that may contribute to joint degeneration are poorly understood. Proteomic analysis of synovial fluid in patients with shoulder instability may improve our understanding of proteins that are shed into shoulder synovial fluid after an injury. Hypothesis: Injury-specific factors such as the direction of instability and the severity of glenoid and humeral bone loss are associated with the proteome of synovial fluid in patients with shoulder instability. Study Design: Descriptive laboratory study. Methods: Synovial fluid lavage samples were compared between patients with anterior (n = 12) and posterior (n = 8) instability and those without instability (n = 5). Synovial proteins were identified with liquid chromatography–tandem mass spectrometry. Orthogonal validation of protein targets found to be significant on tandem mass spectrometry was performed in a separate set of prospective patients with Western blotting. Data were processed and analyzed, and P values were adjusted with the Benjamini-Hochberg method for multiple comparisons. Results: A total of 25 patients were included. Tandem mass spectrometry identified 720 protein groups in synovial fluid of patients with shoulder instability. There were 4 synovial proteins that were significantly expressed in patients with anterior instability relative to posterior instability: periostin (POSTN) (adjusted P value = .03; log fold change [logFc] = 4.7), transforming growth factor beta–induced protein ig-h3 (adjusted P value = .05; logFc = 1.7), collagen type VI alpha-3 chain (adjusted P value = .04; logFc = 2.6), and coagulation factor V (adjusted P value = .04; logFc = −3.3). Among these targets, POSTN showed a moderate correlation with the Hill-Sachs lesion size (r = 0.7). Prospective validation with Western blotting confirmed a significantly higher level of POSTN in synovial fluid of patients with anterior instability (P = .00025; logFc = 5.1). Conclusion: Proteomic analysis enriched our understanding of proteins that were secreted into shoulder synovial fluid of patients with shoulder instability. The identification of POSTN, a proinflammatory catabolic protein involved with tissue remodeling and repair, as a significant target in anterior shoulder instability is a novel finding. Therefore, further study is warranted to determine the role that POSTN may play in the progression of bone loss and posttraumatic osteoarthritis. Clinical Relevance: Proteomic analysis of synovial fluid in patients with shoulder instability improved our understanding of this abnormality after an injury.

Enhancing pathogen detection and characterization of respiratory microbiome signatures of infection through next-generation sequencing

Next-generation sequencing-based (NGS) methods for pathogen detection, which can capture the entire microbial composition in a clinical sample, are used strategically in parallel to culture-based methods. While NGS-based methods cannot confirm organism viability, culture-based methods can but are limited by the number of culture conditions routinely utilized in clinical microbiology laboratories. The relatively wide scope provided by sequencing compensates for the limited scope of culture-based methods but creates obstacles in distinguishing pathogens from an expected body-site-specific microbiome or benign transient colonizers. This study compares NGS-based methods for pathogen detection in respiratory samples to current diagnostics at Texas Children's Hospital in Houston, Texas. Current diagnostics include various culture-based assays, targeted PCR assays, and reference laboratory-generated sequencing. We hypothesize that employing sample metadata (source, type, etc.) and patient data (clinical history, chronic conditions, etc.) during data analysis will help distinguish pathogens from expected flora in respiratory samples. To develop these methods, 27 respiratory samples with suffcient volume for both routine diagnostic testing and this study were collected. Nucleic acid was extracted using the Qiagen DNeasy PowerSoil Pro Kit after collection, and targeted short-read sequencing (16S, 18S/ITS) was performed. These data were then analyzed using a custom bioinformatic pipeline built around Qiime2-2022. In brief, sequences are denoised and quality filtered using DADA2, an algorithm that uses a statistical model for correcting sequencing errors, after which similar amplicon sequence variants (ASVs) are clustered. Next, a feature table of ASVs that are 100% identical is generated, and taxonomy assigned using a naive Bayes taxonomy classifier against the Silva 13.2 database. Finally, unassigned and low abundance ASVs are removed. When species-level identification couldn't be determined through targeted short-read sequencing, untargeted long-read sequencing was used. In one case, we found that a bronchoalveolar lavage sample from the lung of a patient with a history of recurrent pneumonia contained a singular pathogenic bacterium, H. influenzae. This finding agreed with results obtained through reference laboratory-generated sequencing. In another case, we identified >14 unique bacteria at varying concentrations in a wash sample collected from the lung of an asymptomatic patient during a routine bronchoscopy. These findings, however, were determined to be expected respiratory flora. Our NGS-based methods’ enhanced pathogen detection compared to current diagnostics and discriminates between respiratory flora and active infections. Setting thresholds for pathogen detection, evaluating clinical relevance in the context of body site and presentation, and deploying additional molecular techniques when necessary are crucial steps to increase the accuracy of pathogen detection in the clinic. This study was supported by the Texas Children's Hospital Department of Pathology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prevalence of tuberculosis in children using Gene Xpert on gastric lavage samples

Prevalence of tuberculosis in children using Gene Xpert on gastric lavage samples

Excess airway iron during severe SARS-CoV-2 infection impairs neutrophil antifungal activity enabling 2° mold infection

Abstract COVID-19-Associated Pulmonary Aspergillosis (CAPA) is an invasive 2° mold infection caused by Aspergillus species affecting ~5% of patients with severe SARS-CoV-2 (SARS-2) infection. In this study, we hypothesized that excess airway iron released during SARS-2 infection promotes 2° infection via enhancement of fungal growth and impairment of neutrophil antifungal activity. We show that SARS-2 bronchoaveolar lavage samples (n=48) have higher levels of total iron, ferric iron, and oxyhemoglobin relative to bacterial pneumonia (n=40). Likewise, FFPE lung tissues from SARS-2 infection exhibited increased extracellular ferric iron, ferrous iron, ferritin, and hemoglobin. We show that both iron and hemoglobin enhance the growth rate of Aspergillus fumigatus via siderophore and reductive iron assimilation dependent mechanism. Utilizing lung epithelial cell transmigration model, we show that iron enhances neutrophil intracellular ROS production resulting in enhanced conidiacidal activity. In contrast, transmigrated neutrophils exposed to both heme and hemoglobin exhibit decreased ROS production and NET formation with significant impairment in hyphal killing. When both neutrophils and fungi are co-cultured with iron, heme, or hemoglobin, fungi consistently win the battle characterized by uncontrolled hyphal proliferation. These data identify both microbial and host mechanisms which may be targeted pharmacologically to mitigate the iron-dependent growth of 2° mold infection.

Detection of Pathogenic Leptospira in Captive Chelonians (Kinosternon scorpioides-Linnaeus, 1766) in the Brazilian Amazon.

Leptospirosis is a zoonosis of great importance for One Health. In this context, the Amazonian biome may harbor numerous hosts for Leptospira spp. that contribute to the maintenance of the pathogen in the environment. Some reptiles, such as chelonians, have been little studied in terms of their involvement with leptospires. The objective of this study was to detect Leptospira spp. DNA in Kinosternon scorpioides turtles kept in captivity in a region of the Brazilian Amazon. A total of 147 samples of blood (n = 40), cloacal fluid (n = 27), cloacal lavage (n = 40), and stomach (n = 40) were collected from 40 chelonians. After DNA extraction, the samples were subjected to amplification of a 331 base pair product of the 16S rRNA gene using the Lep1 and Lep2 primers. PCR products were Sanger sequenced, assembled, and subjected to online blast search and phylogenetic analysis. Of the animals tested, 40% (16/40, 95% confidence interval [CI]: 25-55) had at least one or two samples positive for Leptospira spp. Considering the total number of samples collected, 12.93% (19/147) were positive, being blood clots (27.5%; 11/40), followed by cloacal washings (10%; 4/40), cloacal fluid (11.11%; 3/27) and gastric washings (2.5%; 1/40). Of these, 11 samples were sequenced and showed 99% to 100% identity with Leptospira interrogans sequences, which was confirmed by phylogenetic analysis. This is the first study to detect pathogenic Leptospira DNA in chelonians in a region of the Brazilian Amazon. It has been concluded that K. scorpioides turtles in captivity have been exposed to pathogenic Leptospira.

Improving diagnostic accuracy of identifying gastric cancer patients with peritoneal metastases: tumor-guided cell-free DNA analysis of peritoneal fluid.

Detection of peritoneal dissemination (PD) in gastric cancer (GC) patients remains challenging. The feasibility of tumor-guided cell-free DNA (cfDNA) detection in prospectively collected peritoneal fluid (ascites and peritoneal lavage) was investigated and compared to conventional cytology in 28 patients. Besides conventional cytology, next generation sequencing was performed on primary tumor DNA and cell-free DNA from peritoneal fluid. Patients were retrospectively grouped into: a positive group (with PD) and a negative group (without PD). Detectable mutations were found in the primary tumor of 68% (n = 19). Sensitivity of PD detection by tumor-guided cfDNA analysis was 91%, compared to 64% by conventional cytology. Within the positive group (n = 11), tumor-guided cfDNA was detected in all patients with ascites samples (4/4, 100%) and in 86% (6/7) of the lavage samples, opposed to 4/4 (100%) patients with ascites and 43% (3/7) with lavage by conventional cytology. Within the negative group (n = 8), conventional cytology was negative for all samples. In two patients, tumor-guided cfDNA was detected in peritoneal lavage fluid. Interestingly, these 2 patients developed PD within 6 months, suggesting a prognostic value of tumor-guided cfDNA detection. This study showed that tumor-guided cfDNA detection in peritoneal fluids of GC patients is feasible and superior to conventional cytology in detecting PD.

Clinical and morphological features of lung injury long-term after SARS-CoV-2 recovery

To study the clinical and histological profile of lung tissue in patients with persistent pulmonary disease, respiratory symptoms and CT findings after SARS-CoV-2 infection. The study included 15 patients (7 females and 8 males) with a mean age of 57.7 years. All patients underwent laboratory tests, chest computed tomography, echocardiography, and pulmonary function tests. Pulmonary tissue and bronchoalveolar lavage samples were obtained by fibrobronchoscopy, transbronchial forceps (2 patients), and lung cryobiopsy (11 patients); open biopsy was performed in 2 patients. Cellular composition, herpesvirus DNA, SARS-CoV-2, Mycobacterium tuberculosis complex, galactomannan optical density index, and bacterial and fungal microflora growth were determined in bronchoalveolar lavage. SARS-CoV-2 was also identified in samples from the nasal mucosa, throat and feces using a polymerase chain reaction. The results showed no true pulmonary fibrosis in patients recovered from SARS-CoV-2 infection with persistent respiratory symptoms, functional impairment, and CT findings after SARS-CoV-2 infection. The observed changes comply with the current and/or resolving infection and inflammatory process. Thus, no true pulmonary fibrosis was found in patients after SARS-CoV-2 infection with persistent respiratory symptoms, functional impairment, and CT findings. The observed changes comply with the current and/or resolving infection and inflammatory process.

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial

Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

Herpesviridae and Atypical Bacteria Co-Detections in Lower Respiratory Tract Samples of SARS-CoV-2-Positive Patients Admitted to an Intensive Care Unit.

Shortly after the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cases of viral, bacterial, and fungal coinfections in hospitalized patients became evident. This retrospective study investigates the prevalence of multiple pathogen co-detections in 1472 lower respiratory tract (LRT) samples from 229 SARS-CoV-2-positive patients treated in the largest intensive care unit (ICU) in Slovenia. In addition to SARS-CoV-2, (rt)RT-PCR tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), and atypical bacteria: Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila/spp. At least one co-detection was observed in 89.1% of patients. EBV, HSV-1, and CMV were the most common, with 74.7%, 58.1%, and 38.0% of positive patients, respectively. The median detection time of EBV, HSV-1, and CMV after initial SARS-CoV-2 confirmation was 11 to 20 days. Bronchoalveolar lavage (BAL) and tracheal aspirate (TA) samples showed equivalent performance for the detection of EBV, CMV, and HSV-1 in patients with both available samples. Our results indicate that SARS-CoV-2 infection could be a risk factor for latent herpesvirus reactivation, especially HSV-1, EBV, and CMV. However, additional studies are needed to elucidate the clinical importance of these findings.

Galactomannan Antigen Assay for the Diagnosis of Invasive Aspergillosis: A Study from a Tertiary Care Hospital of Central India

Invasive Aspergillosis (IA) is a severe and fatal infection, especially in immunocompromised patients. Galactomannan is a polysaccharide antigen present in the cell wall of Aspergillus species, which is secreted into the blood and other body fluids during hyphal growth. Therefore, detecting galactomannan antigen is very useful in diagnosing IA, along with clinical features and radiological findings. The study period was one year (January 2022 to December 2022). The data was collected retrospectively from the medical records and case sheets of all clinically suspected invasive aspergillosis patients. Galactomannan antigen assay was performed using an FDA-approved Platelia Aspergillus EIA test kit, and results were interpreted according to the manufacturer’s instructions (Cut off > 0.5). A total of 236 clinically suspected Invasive Aspergillosis cases were enrolled in the study. Galactomannan positivity was predominantly seen in patients aged 40 – 60 years, with male preponderance. Of 236 patients, 14.40% were immunocompromised, and 85.59% were immunocompetent. According to EORTC/MSG definitions, we got one proven IA case, 21 probable cases (8.89%), and nine (3.81%) possible cases. In immunocompetent individuals also, we observed 48.72% (115/236) galactomannan positivity, especially in old Pulmonary Tuberculosis (PTB) patients. Galactomannan positivity was higher in Broncho alveolar lavage (BAL) samples (n=70, 85.36%) than in serum samples (n=77, 46.67%). We found culture positivity of 14.06%, with Aspergillus fumigatus being the commonest isolate, followed by Aspergillus flavus. There is increased positivity of galactomannan in BAL samples compared to serum; hence BAL is a better specimen for diagnosis of Invasive Pulmonary Aspergillosis (IPA).

Prospective and systematic screening for invasive aspergillosis in the ICU during the COVID-19 pandemic, a proof of principle for future pandemics.

The diagnostic performance of a prospective, systematic screening strategy for COVID-19 associated pulmonary aspergillosis (CAPA) during the COVID-19 pandemic was investigated. Patients with COVID-19 admitted to the ICU were screened for CAPA twice weekly by collection of tracheal aspirate (TA) for Aspergillus culture and PCR. Subsequently, bronchoalveolar lavage (BAL) sampling was performed in patients with positive screening results and clinical suspicion of infection. Patient data were collected from April 2020-February 2022. Patients were classified according to 2020 ECMM/ISHAM consensus criteria. In total, 126/370 (34%) patients were positive in screening and CAPA frequency was 52/370 (14%) (including 13 patients negative in screening). CAPA was confirmed in 32/43 (74%) screening positive patients who underwent BAL sampling. ICU mortality was 62% in patients with positive screening and confirmed CAPA, and 31% in CAPA cases who were screening negative. The sensitivity, specificity, positive and negative predictive value (PPV & NPV) of screening for CAPA were 0.71, 0.73, 0.27, and 0.95, respectively. The PPV was higher if screening was culture positive compared to PCR positive only, 0.42 and 0.12 respectively. CAPA was confirmed in 74% of screening positive patients, and culture of TA had a better diagnostic performance than PCR. Positive screening along with clinical manifestations appeared to be a good indication for BAL sampling since diagnosis of CAPA was confirmed in most of these patients. Prospective, systematic screening allowed to quickly gain insight into the epidemiology of fungal superinfections during the pandemic and could be applicable for future pandemics.

Experience of flexible bronchoscopy in the pediatric pulmonary diseases clinic.

Flexible bronchoscopy is a valuable method in the diagnosis and treatment of respiratory tract diseases in children. This study aimed to examine the indications for and results of flexible bronchoscopy in children. The study included patients aged 0-18 years who underwent flexible bronchoscopy between 1 January 2017 and 31 December 2022. The patients were evaluated for demographic characteristics, indications for bronchoscopy, comorbidities, bronchoscopy findings, and the results of bronchoalveolar lavage. During the defined study period, a total of 410 flexible bronchoscopy procedures were performed. 51.9% of the patient population were male, and 48.1% were female, with a mean age of 96.93 ± 63.45 months. The most common indication for flexible bronchoscopy was recurrent lower respiratory tract infection (26.8%), followed by chronic cough (19.1%). The bronchoalveolar lavage culture results showed that the most commonly isolated microorganisms were H. influenzae non-type b (7.8%) followed by M. catarrhalis (7.3%). Mucus obstruction and secretion (33.0%) constituted the most common bronchoscopic findings, while the flexible bronchoscopy examination was normal in 27% of patients. No serious complications occurred in any patient during or after the procedure. The results of this study demonstrated that the most common indication for flexible bronchoscopy was recurrent lower respiratory tract infection and the most common bronchoscopy finding was purulent secretion with mucus obstruction. Flexible bronchoscopy is an important diagnostic and treatment tool for patients with recurrent respiratory symptoms. It is a highly valuable method as it enables direct visualization of the airways and facilitates the collection of bronchoalveolar lavage samples.

Association of clinical respiratory disease signs and lower respiratory tract bacterial pathogens with systemic inflammatory response in preweaning dairy calves

This study investigated the potential associations between 3 acute phase proteins (APP)-haptoglobin, serum amyloid A-and fibrinogen, clinical signs of respiratory disease, and the presence of bacterial pathogens in the lower respiratory tract (LRT) of preweaning dairy calves. This cross-sectional study included 150 preweaning calves (2-86 d old) from 15 large dairy herds in Estonia. Tracheobronchial lavage, blood, and fecal samples were collected from 5 calves showing clinical signs indicative of LRT disease, and samples from 5 calves without clinical signs of LRT disease per herd. All samples collected from these calves were analyzed for concentrations of systemic APP, LRT bacteria, and intestinal pathogens. Heifer blood and bulk tank milk samples were collected for the detection of disease-specific antibodies against bovine herpesvirus 1, bovine viral diarrhea virus, bovine respiratory syncytial virus, and Mycoplasma bovis. Mixed-effects linear regression models were used to analyze the associations of clinical respiratory disease signs and LRT bacteria with APP. Increased plasma fibrinogen concentrations in calves were associated with higher rectal temperature (>39.5°C), increased respiratory rate (>50 breaths/min), and coughing. Increased serum amyloid A concentrations were associated with higher rectal temperature (>39.5°C) and respiratory rate between 40 and 50 breaths/min. Calves with the presence of fecal Cryptosporidium spp. and rectal temperature of 39°C and above had increased serum haptoglobin concentrations. Increased fibrinogen concentrations were associated with the presence of Pasteurella multocida in the calf LRT, whereas increased concentrations of fibrinogen and serum amyloid A were associated with the presence of Trueperella pyogenes. In conclusion, APP showed variable associations with clinical signs of respiratory disease and LRT bacteria. Plasma fibrinogen concentration could be used as a complementary calf-side test to assess systemic inflammation caused by LRT bacteria such as Pasteurella multocida and Trueperella pyogenes in preweaning dairy calves.

Gut microbiome signature and nasal lavage inflammatory markers in young people with asthma

BackgroundAsthma is a complex disease and a severe global public health problem resulting from interactions between genetic background and environmental exposures. It has been suggested that the gut microbiota may be related to asthma development, however, such relationships needs further investigation. ObjectiveThis study aimed to characterize the gut microbiota as well as the nasal lavage cytokine profile of asthmatic and non-asthmatic individuals. MethodsStool and nasal lavage samples were collected from 29 children and adolescents with Type 2-asthma (defined as asthma with >2.5% eosinophils in sputum, and a positive skin prick test) and 28 children without asthma in Brazil. Amplicon sequencing of the stool bacterial V4 region of the 16S rRNA gene was performed using Illumina MiSeq. Microbiota analysis was performed using QIIME2 and PICRUST. Type 2-asthma phenotype was characterized by high sputum eosinophil counts and positive skin prick tests for house dust mites, cockroaches, cat or dog dander. The nasal immune marker profile was assessed using a customized multiplex panel. ResultsThe stool microbiota differed significantly between asthmatic and non-asthmatic participants (p=0.001). Bacteroides was more abundant in participants with asthma (p<0.05), while Prevotella was more abundant in non-asthmatics (p<0.05). In asthmatics, the relative abundance of Bacteroides correlated with IL-4 concentration in nasal lavage. Inference of microbiota functional capacity identified differential fatty acid biosynthesis in asthmatics compared to non-asthmatics. ConclusionThe stool microbiota differed between asthmatics and non-asthmatics in young people in Brazil. Asthma was associated with higher Bacteroides which correlated with nasal IL-4 concentration.

NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

Non-Tuberculous Mycobacterial isolates from Panama: A retrospective 5-year analysis (2017-2021)

BackgroundThe genus Mycobacterium includes well-known bacteria such as M. tuberculosis causing tuberculosis and M. leprae causing leprosy. Additionally, various species collectively termed non-tuberculous mycobacteria (NTM) can cause infections in humans and animals, affecting individuals across all age groups and health conditions. However, information on NTM infection prevalence in Panama is limited. MethodsThis study conducted a retrospective analysis of clinical records from 2017 to 2021, specifically focusing on patients with NTM isolates. Data were categorized by variables like sex, age, HIV status, and sample source. ResultsAmong the 4430 clinical records analyzed, 698 were linked to patients with NTM isolates. Of these patients, 397 were male, and 301 were female. Most female patients with NTM isolates (n = 190) were aged >45 to 85 years, while most male patients (n = 334) fell in the >25 to 75 years age group. A noteworthy proportion of male patients (n = 65) were aged 25–35 years. A significant age difference between male (median [min-max] = 53 years [3–90]) and female (median [61 years [6–94]) patients was observed (p < 0.001). Regarding HIV status, 77 positive individuals were male, and 19 were female (p < 0.001). Most samples (n = 566) were sputum samples, with additional pulmonary-associated samples such as broncho-alveolar lavage, tracheal secretions, and pleural fluid samples. Among extrapulmonary isolates (n = 48), sources included catheter secretions, intracellular fluids, peritoneal fluid, blood cultures, cerebrospinal fluid, bone marrow samples, and capillary transplant lesions. Specifically, the analysis identified the pathogenic microorganisms responsible for mycobacteriosis in Panama during the specific period 2017–2021, as M. fortuitum (34.4%), M. intracellulare (20.06%), and M. abscessus (13.75%), respectively. ConclusionsThis study highlights the growing public health concern of NTM infections in Panama. The research provides valuable insights into the prevalence and distribution of NTM species in the country, offering a foundation for the development and implementation of effective prevention and control strategies for NTM infections in Panama.

МИКРОБИОЦЕНОЗЫ ПРИ ОСТРОЙ КАТАРАЛЬНОЙ БРОНХОПНЕВМОНИИ ТЕЛЯТ

The purpose of the study is to conduct intravital diagnostics of calves with acute catarrhal bronchopneumonia using bronchoalveolar lavage samples and a detailed study of microbiocenoses from the collected contents. Objectives: development of a technique for intravital collection of bronchoalveolar lavage in calves with bronchopneumonia; study of the microbial landscape in calves with bronchopneumonia, characteristics of isolated microorganisms and their associations. The object of the study is calves aged 1–3 months, sick with acute catarrhal bronchopneumonia (n = 37). Pathological bronchoalveolar lavage was collected transnasally from the tracheal bifurcation area using sterile silicone catheters into sterile test tubes. Bacteriological studies were conducted using generally accepted methods at the Scientific and Educational Resource Center (SERC) Pharmacy of the People's Friendship University of Russia. As a result of the studies, 115 microorganisms of 13 species, classified into 9 genera, were isolated. The majority of isolates – 71 (61.7 %) were classified as gram-negative microorganisms. Staphylococcus aureus, Mannheimia haemolytica, Escherichia coli, Pasteurella multocida, Klebsiella pneumoniae, Streptococcus pyogenes and Klebsiella ozaenae were most often isolated from pathological material in cases of bronchopneumonia in calves. Isolates of E. coli were most often represented by O8, O26 and O111 serotypes. Of the 115 microorganisms that initiated bronchopneumonia in calves, the majority – 75 (65.2 %) cultures – had pathogenic properties. Retrospective analysis of microbial associations showed that most often the development of acute catarrhal bronchopneumonia in calves is caused by associations of opportunistic microorganisms, which included from 2 to 5 pathogens. Moreover, associations that included three members were most often isolated – 21 (56.8 %) and two members – 8 (21.6 %). Thus, in acute catarrhal bronchopneumonia of calves, microbiocenoses are formed in the inflammation focus, which can include various variations of opportunistic microorganisms.

Identification of the major immune differences in severe asthmatic children according to their atopic dermatitis status

Severe asthma (SA) affects 2% to 5% of asthmatic children. Atopic dermatitis can affect up to 34% of children with SA (cwSA). Atopic dermatitis and asthma share common genetic and immunological features. However, not all children with SA suffer from AD, and it remains unclear whether the overall immune profiles of these children are similar. In this study, seventeen cwSA (9.8 [7.1–13.2] years; seven with and ten without AD) were enrolled. Bronchoalveolar lavage (BAL) and blood samples were collected from these patients. Seventy-three cytokines/chemokines and distinct immune T cell populations were evaluated in blood and BAL. We found that BAL and blood immune profiles of cwSA with and without AD were globally similar. However, specific differences were observed, namely lower frequency of Tc2, Th17 and IL-17-producing mucosal associated invariant T (MAIT-17) cells and higher CD8/CD4 ratio and IL-22 concentrations in BAL and of CCL19 concentrations in plasma from cwSA with AD. Further, in contrast with cwSA without AD, we found a positive correlation between a set of plasma cytokines and almost all cytokines in BAL in cwSA with AD. In conclusion, this study shows the major immune differences between cwSA with and without AD in BAL and blood suggesting that distinct endotypes may be implicated in the inflammatory responses observed in these pediatric patients.

Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.