Yeast species in the respiratory samples of COVID-19 patients; molecular tracking of Candida auris.

Introduction: Although the existence of Candida species in the respiratory tract is often considered commensal, it is crucial to recognize the significance of Candida colonization in immunocompromised or COVID-19 patients. The emergence of Candida auris as an emerging pathogen further emphasizes the importance of monitoring yeast infection/colonization, particularly in COVID-19 patients. Methods: In this study, respiratory samples mainly from COVID-19 patients, primarily those suspected of having a fungal infection, were cultured on Sabouraud dextrose agar plates and the yeast colonies were identified using a two-step multiplex PCR method. The samples suspected of C. auris underwent specific nested PCR followed by sequence analysis. Results: A total of 199 respiratory samples were collected from 73 women and 126 men, ranging in age from 1.6 to 88 years. Among the patients, 141 had COVID-19, 32 had cancer, 5 were hospitalized in ICU, 2 had chronic obstructive pulmonary disease)COPD(, and others were patients with combination diseases. From these samples, a total of 334 yeast strains were identified. C. albicans (n=132, 39.52%) was the most common species, followed by C. tropicalis (n=67, 20%), C. glabrata (n=56, 16.76%), C. krusei (n=18, 5.4%), C. parapsilosis (n=17, 5.08%), Saccharomyces cerevisiae (n=10, 3%), C. kefyr (n=9, 2.6%), C. dubliniensis (n=7, 2.1%), C. lusitaniae (n=5, 1.5%), C. auris (n=3, 0.9%), C. guilliermondii (n=2, 0.6%), C. rugosa (n=1, 0.3%), C. intermedia (n=1, 0.3%), and Trichosporon spp. (n=1, 0.3%). C. auris was detected in a patient in ICU and two COVID-19 patients. While its presence was confirmed through sequence analysis, our extensive efforts to isolate C. auris were unsuccessful. Conclusion: While C. albicans colonization remains prevalent, our study found no evidence of Candida lung infection. Since the role of Candida colonization in airway secretions remains ambiguous due to limited research, further studies are imperative to shed light on this matter.

Rapid Diagnosis of Pneumocystis jirovecii Pneumonia and Respiratory Tract Colonization by Next-Generation Sequencing.

OBJECTIVES: To describe the epidemiology of Pneumocystis jirovecii pneumonia and colonization diagnosed by next-generation sequencing (NGS) and explore the usefulness of the number of P. jirovecii sequence reads for the diagnosis of P. jirovecii pneumonia. METHODS: We examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics. RESULTS: Among 285 respiratory samples collected over a 12-month period (January to December 2022), P. jirovecii sequences were detected in 56 samples from 53 patients. Fifty (94.3%) of the 53 patients were HIV-negative. Following our case definitions, 37 (69.8%) and 16 (30.2%) of the 53 patients had P. jirovecii infection and colonization respectively. P. jirovecii infection was associated with presence of underlying disease with immunosuppression (94.6% vs 18.8%, P < 0.05), positive serum 1,3-ß-D-glucan (41.2% vs 0%, P < 0.01) and higher number of P. jirovecii sequence reads (P < 0.005). In contrast, P. jirovecii colonization was associated with the male sex (93.8% vs 54.1%, P < 0.01), another definitive infectious disease diagnosis of the respiratory tract (43.8% vs 2.7%, P < 0.001) and higher survival (100% vs 67.6%, P < 0.01). Although P. jirovecii pneumonia was associated with higher number of P. jirovecii reads in respiratory samples, only a sensitivity of 82.14% and a specificity of 68.75% could be achieved. CONCLUSION: Detection of P. jirovecii sequences in respiratory samples has to be interpreted discreetly. A combination of clinical, radiological and laboratory findings is still the most crucial in determining whether a particular case is genuine P. jirovecii pneumonia.

The changing epidemiology of pulmonary infection in children and adolescents with cystic fibrosis: an 18-year experience.

The impact of evolving treatment regimens, airway clearance strategies, and antibiotic combinations on the incidence and prevalence of respiratory infection in cystic fibrosis (CF) in children and adolescents remains unclear. The incidence, prevalence, and prescription trends from 2002 to 2019 with 18,339 airway samples were analysed. Staphylococcus aureus [- 3.86% (95% CI - 5.28-2.43)] showed the largest annual decline in incidence, followed by Haemophilus influenzae [- 3.46% (95% CI - 4.95-1.96)] and Pseudomonas aeruginosa [- 2.80%95% CI (- 4.26-1.34)]. Non-tuberculous mycobacteria and Burkholderia cepacia showed a non-significant increase in incidence. A similar pattern of change in prevalence was observed. No change in trend was observed in infants < 2 years of age. The mean age of the first isolation of S. aureus (p < 0.001), P. aeruginosa (p < 0.001), H. influenza (p < 0.001), Serratia marcescens (p = 0.006) and Aspergillus fumigatus (p = 0.02) have increased. Nebulised amikacin (+ 3.09 ± 2.24 prescription/year, p = 0.003) and colistin (+ 1.95 ± 0.3 prescriptions/year, p = 0.032) were increasingly prescribed, while tobramycin (- 8.46 ± 4.7 prescriptions/year, p < 0.001) showed a decrease in prescription. Dornase alfa and hypertonic saline nebulisation prescription increased by 16.74 ± 4.1 prescriptions/year and 24 ± 4.6 prescriptions/year (p < 0.001). There is a shift in CF among respiratory pathogens and prescriptions which reflects the evolution of cystic fibrosis treatment strategies over time.

Comparison of PAS and adenoids in patients with and without maxillary micrognathia before orthodontic treatment.

OBJECTIVE: Craniofacial anomalies are widely discussed as predisposing factors of breathing disorders. Since many more cofactors exist, this study investigated the association between maxillary micrognathia and morphological changes of posterior airway space and adenoids in these patients. MATERIAL AND METHODS: Cephalometric radiographs of n = 73 patients were used for data acquisition. The patients were divided into two groups according to certain skeletal characteristics: maxillary micrognathia (n = 34, 16 female, 18 male; mean age 10.55 ± 3.03 years; defined by a SNA angle < 79°) and maxillary eugnathia (n = 39, 19 female, 20 male; mean age 10.93 ± 3.26 years; defined by a SNA angle > 79°). The evaluation included established procedures for measurements of the maxilla, posterior airway space and adenoids. Statistics included Kolmogorov-Smirnov-, T- and Mann-Whitney-U-Tests for the radiographs. The level of significance was set at p < 0.05. RESULTS: The cephalometric analysis showed differences in the superior posterior face height and the depth of the posterior airway space at palatal level among the two groups. The depth of the posterior airway space at mandibular level was the same for both groups, just as the size of the area taken by adenoids in the nasopharynx. CONCLUSIONS: Skeletal anomalies affect the dimension of the posterior airway space. There were differences among the subjects with maxillary micrognathia and these with a normal maxilla. However, the maxilla was only assessed in the sagittal direction, not in the transverse. This study showed that the morphology of the maxilla relates to the posterior airway space whereas the adenoids seem not to be affected. CLINICAL RELEVANCE: Maxillary micrognathia is significantly associated with a smaller depth of the posterior airway space at the palatal level compared to patients with maxillary eugnathia.

Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®â€¢GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®â€¢GUT will yield more representative microbial communities.

Epithelial cells crowded out in asthma.

Bronchoconstriction causes epithelial cell extrusion that promotes airway inflammation.

The Roles of Neuropeptide Y in Respiratory Disease Pathogenesis via the Airway Immune Response.

The lungs are very complex organs, and the respiratory system performs the dual roles of repairing tissue while protecting against infection from various environmental stimuli. Persistent external irritation disrupts the immune responses of tissues and cells in the respiratory system, ultimately leading to respiratory disease. Neuropeptide Y (NPY) is a 36-amino-acid polypeptide and a neurotransmitter that regulates homeostasis. The NPY receptor is a seven-transmembrane-domain G-protein-coupled receptor with six subtypes (Y1, Y2, Y3, Y4, Y5, and Y6). Of these receptors, Y1, Y2, Y4, and Y5 are functional in humans, and Y1 plays important roles in the immune responses of many organs, including the respiratory system. NPY and the Y1 receptor have critical roles in the pathogenesis of asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis. The effects of NPY on the airway immune response and pathogenesis differ among respiratory diseases. This review focuses on the involvement of NPY in the airway immune response and pathogenesis of various respiratory diseases.

Meta-analysis of the human upper respiratory tract microbiome reveals robust taxonomic associations with health and disease.

BACKGROUND: The human upper respiratory tract (URT) microbiome, like the gut microbiome, varies across individuals and between health and disease states. However, study-to-study heterogeneity in reported case-control results has made the identification of consistent and generalizable URT-disease associations difficult. RESULTS: In order to address this issue, we assembled 26 independent 16S rRNA gene amplicon sequencing data sets from case-control URT studies, with approximately 2-3 studies per respiratory condition and ten distinct conditions covering common chronic and acute respiratory diseases. We leveraged the healthy control data across studies to investigate URT associations with age, sex, and geographic location, in order to isolate these associations from health and disease states. CONCLUSIONS: We found several robust genus-level associations, across multiple independent studies, with either health or disease status. We identified disease associations specific to a particular respiratory condition and associations general to all conditions. Ultimately, we reveal robust associations between the URT microbiome, health, and disease, which hold across multiple studies and can help guide follow-up work on potential URT microbiome diagnostics and therapeutics.

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

The characteristics of microbiome in the upper respiratory tract of COVID-19 patients.

BACKGROUND: Co-infection with other pathogens in coronavirus disease 2019 (COVID-19) patients exacerbates disease severity and impacts patient prognosis. Clarifying the exact pathogens co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is premise of the precise treatment for COVID-19 patients. METHODS: Sputum samples were collected from 17 patients in the COVID-19 positive group and 18 patients in the COVID-19 negative group. DNA extraction was performed to obtain the total DNA. Sequencing analysis using 16S and ITS rRNA gene was carried out to analyze the composition of bacterial and fungal communities. Meanwhile, all the samples were inoculated for culture. RESULTS: We did not observe significant differences in bacterial composition between the COVID-19 positive and negative groups. However, a significantly higher abundance of Candida albicans was observed in the upper respiratory tract samples from the COVID-19 positive group compared to the COVID-19 negative group. Moreover, the Candida albicans strains isolated from COVID-19 positive group exhibited impaired secretion of aspartyl proteinases. CONCLUSION: COVID-19 positive patients demonstrate a notable increase in the abundance of Candida albicans, along with a decrease in the levels of aspartyl proteinases, indicating the alteration of microbiota composition of upper respiratory tract.

Assessment of maximal inspiratory and expiratory pressures in skeletal Class II patients with different growth patterns.

OBJECTIVES: To evaluate maximal inspiratory (MIP) and expiratory (MEP) pressures, which are reflective of respiratory muscle strength, in skeletal Class II patients with different growth patterns (horizontal, average, and vertical) and to correlate those with airway dimension. MATERIALS AND METHODS: Patients with a Class II skeletal base seeking orthodontic treatment were assigned to the following groups: average, horizontal, and vertical growth pattern. The control group (n = 14) comprised patients with a Class I skeletal base and average growth pattern. Airway dimensions were obtained using cone-beam computed tomography scans, and a spirometer with a pressure transducer was used for assessment of MIP and MEP. Routine spirometry for assessment of lung function was also performed. RESULTS: No significant differences were found in maximal inspiratory and expiratory pressures for the study groups in comparison with the control group. Class I patients had significantly greater oropharyngeal and nasopharyngeal airway volumes compared with the study groups. No significant difference in minimal cross-section area of the airway was observed among groups. A weak positive correlation between maximal inspiratory pressure and airway volume was observed. CONCLUSIONS: Although Class I patients displayed significantly greater oropharyngeal and nasopharyngeal airway volumes, there was no significant difference in respiratory muscle strength or airway function between Class II patients with different growth patterns and the Class I control group. The findings underscore the significance of exploring factors beyond craniofacial growth patterns that may contribute to sleep-related breathing disorders.

Lipid nanoparticles for local delivery of mRNA to the respiratory tract: Effect of PEG-lipid content and administration route.

Design of inhalable mRNA therapeutics is promising because local administration in the respiratory tract is minimally invasive and induces a local response. However, several challenges related to administration via inhalation and respiratory tract barriers have so far prevented the progress of inhaled mRNA therapeutics. Here, we investigated factors of importance for lipid nanoparticle (LNP)-mediated delivery of mRNA to the respiratory tract. We hypothesized that: (i) the PEG-lipid content is important for providing colloidal stability during aerosolization and for mucosal delivery, (ii) the PEG-lipid contentinfluences the expression of mRNA-encoded protein in the lungs, and (iii) the route of administration (nasal versus pulmonary) affects mRNA delivery in the lungs. In this study, we aimed to optimize the PEG-lipid content for mucosal delivery and to investigatethe effect of administration route on the kinetics of protein expression. Our results show that increasing the PEG-lipid content improves the colloidal stability during the aerosolization process, but has a negative impact on the transfection efficiencyin vitro. The kinetics of protein expressionin vivois dependent on the route of administration, and we found that pulmonaryadministration of mRNA-LNPs to mice results inmore durable protein expression than nasaladministration. These results demonstrate that the design of the delivery system and the route of administration are importantfor achieving high mRNA transfection efficiency in the respiratory tract.

Surveillance of infections of surgical sites and lower respiratory tracts should be combined: experiences from the German surveillance module for operated patients (OP-KISS), 2018 to 2022.

BackgroundSurveillance of lower respiratory tract infections (LRTI) of operated patients conventionally focuses on intubated patients in intensive care units (ICU). Post-operative immobilisation increases the risk of LRTI not associated with ventilators. Operated patients, however, have thus far not been a primary target for LRTI surveillance.AimWe aimed to describe the applied LRTI surveillance method in the German surveillance module for operated patients (OP-KISS) and to report data between 2018 and 2022.MethodsSurveillance of LRTI can be performed voluntarily in addition to surgical site infection (SSI) surveillance in OP-KISS. We calculated LRTI rates per 100 operations for all procedures combined, as well as for individual surgical groups and procedures. Additionally, a combined post-operative infection rate (SSI and LRTI) was calculated.ResultsSurveillance of LRTI was performed in 4% of all participating OP-KISS departments and for 2% (23,239 of 1,332,438) of all procedures in the OP-KISS database. The pooled LRTI rate was 0.9 per 100 operations, with marked differences between different types of surgery (3.6 for lobectomies, 0.1 for traumatology and orthopaedics). The share of LRTI among all post-operative infections was highly variable. For lobectomies, the LRTI rate was higher than the SSI rate (3.6 vs 1.5 per 100 operations).ConclusionSurveillance of post-operative LRTI is not yet widely adopted by German hospitals. Based on the data in this study, lobectomies represent a prime target for post-operative LRTI surveillance.

SARS-CoV-2 infection increases airway bleeding risk in patients after tracheostomies.

BACKGROUND: Airway bleeding events are a rare incident in SARS-CoV-2-infected patients after tracheostomies. We aimed to explore the correlation between airway bleeding and SARS-CoV-2 infection and evaluate the consistency of SARS-CoV-2 RNA test results in the upper and lower airway samples from patients after tracheostomies. METHODS: Forty-four patients after temporary or permanent tracheostomy were divided into a positive group (29 patients) and a negative group (15 patients) based on the SARS-CoV-2 RNA test results of their oropharyngeal swabs. The oropharyngeal and tracheal swabs of the positive group were re-collected for SARS-CoV-2 RNA detection. Demographic and clinical characteristics and airway bleeding events were recorded for all enrolled patients. RESULTS: Airway bleeding was reported in eleven patients of the positive group (11/29), with seven displaying bloody sputum or hemoptysis, and four featuring massive sputum crust formation in the trachea that resulted in dyspnea, and only one patient in the negative group (1/15), with a significant difference in the airway bleeding rate (37.9% vs. 6.7%, p < 0.05). The SARS-CoV-2 RNA test results showed a statistical difference in cycle threshold (Ct) values between oropharyngeal swabs and tracheal swabs (p < 0.05). CONCLUSIONS: After tracheostomies, patients are more susceptible to airway bleeding if they are infected with SARS-CoV-2. The findings signify that in addition to droplet transmission through tracheostoma, SARS-CoV-2 may infect the oropharynx by airborne and close contact transmission, and that given the higher viral load and longer infection time in the trachea, tracheal swabs are more reliable for SARS-CoV-2 detection in these patients.