Wash Samples Research Articles

Analysis of water and aerosol samples of tunnel car washes operated with recycled water for Legionella with culture, qPCR and viability-qPCR

Due to the generation of large quantities of aerosol and the recycling of water, tunnel car washes are discussed as potential sources of legionellosis. Additionally, occupational health and safety aspects are important for tunnel car washes as they are often workplaces.A total of 17 different tunnel car washes were investigated for the presence of Legionella. In the process, 78 water samples and 63 air samples were taken. This comprised samples of municipal and recycled water as well as aerosol from the car wash tunnel and the workplace. The analysis for Legionella included culture method in combination with an immunochromatographic test, qPCR and viability-qPCR. Where possible, Legionella species were identified by sequencing of mip gene.Using the culture method Legionella were detected in 9 of 78 water samples (91 to 10,800 CFU/100 mL). In contrast, quantifiable concentrations of viable Legionella spp. were found in 68 of 78 water samples with viability-qPCR. The median concentration was 9.2 × 105(n = 16) and 7.2 × 105 GU/100 mL (n = 17) for the recycled water from the storage tank and the nozzles in the car wash tunnel. Viable Legionella spp. were detectable in the aerosol at the workplace in 38.1 % of the samples (n = 21). Concentration was between 155 and 3829 GU/m3 (n = 7). L. pneumophila non-serogroup 1 were quantitatively detectable in the recycled water of one car wash, using qPCR methods and culture. Aerosolisation of this species was not detected.The presence of viable Legionella spp. in most water and many aerosol samples as well as the identification of species related to infection suggests that there is a risk of legionellosis through exposure to bioaerosols released from tunnel car washes. Comparison of conventional culture method with qPCR methods showed a considerable underestimation of Legionella concentrations by culture.

Evidence of Site-Specific Mucosal Autoantibody Secretion in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPA) have been detected in sputum and saliva, indicating that anti-modified protein antibodies (AMPA) can be produced at mucosal sites in patients with rheumatoid arthritis (RA). However, the body's largest mucosal compartment, the gut, has not yet been examined. We therefore investigated the presence of several AMPA(ACPA, anti-carbamylated protein antibodies [anti-CarP], and anti-acetylated protein antibodies [AAPA]) at different mucosal sites, including the intestinal tract. Paired fecal/ileal wash, saliva, and serum samples of patients with RA and healthy volunteers were collected in two independent cohorts. Data involving feces were replicated in a third cohort. In these secretions, AMPA were analyzed using in-house enzyme-linked immunosorbent assay with unmodified peptides as control. In fecal samples, total IgA and anti-Escherichia coli IgA were measured. ACPA, anti-CarP, and AAPA IgA were measurable in saliva of seropositive patients with RA (prevalence 9%-40%). No AMPA could be detected in feces. IgA was present because total IgA and anti-E. coli IgA were detectable in feces of ACPA-positive patients with RA and healthy donors. Results were confirmed in another cohort using colonoscopically collected ileal wash samples. Our study shows the presence of ACPA, anti-CarP, and AAPA IgA in saliva of ACPA-seropositive patients with RA. However, no AMPA could be detected in feces/ileal wash samples of these patients, although our assays were able to measure other antigen-specific antibodies. These data suggest that mucosal autoantibody secretion may occur in the oral mucosa of patients with RA, whereas no evidence could be found for this process in the lower gastrointestinal tract.

EDP-938 Has a High Barrier to Resistance in Healthy Adults Experimentally Infected with Respiratory Syncytial Virus.

EDP-938 is an oral once-daily RSV nucleoprotein (N) inhibitor with potent antiviral activity. In a human RSV challenge trial, EDP-938 significantly reduced viral load and symptom severity. During antiviral development, it is critical to understand the propensity for resistance to develop. In vitro studies of EDP-938 suggest a higher barrier to resistance as compared to RSV fusion inhibitors. We evaluated the development of viral resistance to EDP-938 in a human challenge trial. A subset of the 124 participants with RSV infection were chosen for genetic analysis; 159 nasal wash samples from 48 participants were analyzed using next-generation sequencing of the N gene of RSV. Of the 48 participant sampled, 37 were from EDP-938-treated and 11 were placebo-treated participants, representing 45% and 26% of the participants, respectively. The effects of treatment-emergent mutations on viral load, EDP-938 efficacy, and viral fitness were evaluated. Two of the 37 EDP-938-treated participants with samples sequenced had treatment-emergent mutations: N:L139I and N:E112G. From in vitro analysis, N:L139I reduced sensitivity to EDP-938 by approximately 10-fold, while N:E112G had no effect. However, N:L139I was associated with a reduction in viral fitness, suggesting clinical resistance is associated with fitness costs. Neither of these variants were associated with reduced viral clearance. In human RSV infections treated with EDP-938, emergence of RSV variants with reduced sensitivity to EDP-938 occurred in only 1 participant and was associated with reduced viral fitness. EDP-938's high barrier to resistance highlights its robust mechanism of action. NCT03691623.

INFLUENCE OF DISINFECTANTS ON THE TOTAL BACTERIAL CONTAMINATION OF DUCK EGGSHELLS

The role of the total viable microbial count on eggshells is of significant interest, as it relates to egg safety and product shelf life.The aim of this study was to assess the influence of various disinfectants on the overall bacterial contamination of duck eggshells. For this purpose, fresh high-quality duck eggs were obtained from a farm, and solutions of disinfectants were prepared: 6% hydrogen peroxide, 4% calcium hydroxide, 4% sodium carbonate, and 3% acetic acid. When disinfecting duck or goose eggs, the dosage of substances is doubled compared to chicken eggs. The eggs were treated with prepared disinfectant solutions, with varying exposure times. The exposure times for hydrogen peroxide were 4 and 8 minutes, for calcium hypochlorite – 2 and 5 minutes, for sodium carbonate – 10 and 30 minutes, and for acetic acid – 5 and 15 minutes. Samples of washings from the eggshells were collected both before and after disinfection. Dilutions were prepared and subsequently plated on Petri dishes with non-selective media, followed by incubation at 30°C for 72 hours. As a result of colony counting, the number of mesophilic aerobic and facultatively anaerobic microorganisms (CFU/egg) was determined.The results of the study showed a direct correlation between the inactivation of microorganisms and exposure time when using acetic acid, hydrogen peroxide, and calcium hypochlorite. A longer contact period of the eggs with sodium carbonate did not result in a measurable reduction in the number of viable microorganisms. It was also found that 4% calcium hypochlorite could completely inactivate microbes on the eggshell within 5 minutes. With a 2-minute exposure, the total microbial contamination was 9.2*10¹±0.7 CFU/egg.Thus, in this study, solutions of calcium hypochlorite, hydrogen peroxide, and acetic acid demonstrated high bactericidal properties and can be considered effective agents for disinfecting duck eggshells.Most scientific articles are devoted to the study of chicken eggs. In this work, the object of research is duck eggs, the best disinfectants for processing eggshells have been identified, which is an urgent task in poultry farming.The obtained results can help farmers implement more effective strategies for combating foodborne infections.

Vaccine-induced NA immunity decreases viral shedding, but does not disrupt chains of airborne transmission for the 2009 pandemic H1N1 virus in ferrets.

In humans and animal models, immunity against neuraminidase (NA) reduces disease severity and viral replication during influenza infection. However, we have a limited understanding of the impact of NA immunity on viral transmission. Using chains of airborne transmission in ferrets as a strategy to simulate a more natural route of infection, we assessed if vaccine-induced NA immunity could disrupt transmission of the 2009 pandemic H1N1 virus. The 2009 pandemic H1N1 virus transmitted efficiently through chains of transmission in the presence of NA immunity, but NA-vaccinated animals shed significantly less virus and had accelerated viral clearance. To determine if immune pressure led to the generation of escape variants, viruses in ferret nasal wash samples were sequenced, and no mutations in NA were identified. These findings demonstrate that vaccine-induced NA immunity is not sufficient to prevent infection via airborne exposure and onward airborne transmission of the 2009 pandemic H1N1 virus.

Examining intra-host genetic variation of RSV by short read high-throughput sequencing.

Every viral infection entails an evolving population of viral genomes. High-throughput sequencing technologies can be used to characterize such populations, but to date there are few published examples of such work. In addition, mixed sequencing data are sometimes used to infer properties of infecting genomes without discriminating between genome-derived reads and reads from the much more abundant, in the case of a typical active viral infection, transcripts. Here we apply capture probe-based short read high-throughput sequencing to nasal wash samples taken from a previously described group of adult hematopoietic cell transplant (HCT) recipients naturally infected with respiratory syncytial virus (RSV). We separately analyzed reads from genomes and transcripts for the levels and distribution of genetic variation by calculating per position Shannon entropies. Our analysis reveals a low level of genetic variation within the RSV infections analyzed here, but with interesting differences between genomes and transcripts in 1) average per sample Shannon entropies; 2) the genomic distribution of variation 'hotspots'; and 3) the genomic distribution of hotspots encoding alternative amino acids. In all, our results suggest the importance of separately analyzing reads from genomes and transcripts when interpreting high-throughput sequencing data for insight into intra-host viral genome replication, expression, and evolution.

Immune Cell Profiles of Patients with Sickle Cell Disease during Parvovirus B19-Induced Transient Red Cell Aplasia.

Parvovirus B19 frequently infects children and targets cells of the erythroid lineage. Although healthy children rarely suffer severe disease, children with sickle cell disease (SCD) can experience transient red cell aplasia (TRCA), hospitalization, and life-threatening anemia upon first virus exposure. Given that children with SCD can also suffer chronic inflammation and that parvovirus B19 has been associated with autoimmune disease in other patient populations, we asked if parvovirus B19 infections contributed to acute and chronic immune abnormalities in children with SCD. Nineteen hospitalized patients with SCD and parvovirus B19-induced TRCA were evaluated. Blood tests included CBC, flow cytometry, and total antibody isotype analyses. Cytokine/chemokine analyses were performed on nasal wash (NW) samples, representing a common site of viral entry. Unusually high white blood cell count (WBC) and absolute neutrophil count (ANC) values were observed in some patients. A correlation matrix with Day 0 values from the 19 patients then identified two mutually exclusive phenotype clusters. Cluster 1 included WBC, ANC, absolute reticulocyte count (ARC), absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), NW cytokines/chemokines, % naïve cells among B cell and T cell populations, and parvovirus-specific IgG. This cluster was negatively associated with virus load, suggesting a signature of successful adaptive immunity and virus control. Cluster 2 included virus load, % CD38+CD24- cells among CD19+ B cells (termed 'plasmablasts' for simplicity), % HLA-DRlow cells among CD19+ B cells, IgG4, and % memory phenotypes among B cell and T cell populations. Plasmablast percentages correlated negatively with parvovirus-specific IgG, possibly reflecting a non-specific trigger of cell activation. All patients were released from the hospital within 1 week after admission, and the highest WBC and ANC values were eventually reduced. Nonetheless, a concern remained that the acutely abnormal immune profiles caused by parvovirus B19 infections could exacerbate chronic inflammation in some patients. To avoid the numerous sequelae known to affect patients with SCD following hospitalizations with parvovirus B19, rapid development of a parvovirus B19 vaccine is warranted.

Microbiome of esophageal endoscopic wash samples is associated with resident flora in the esophagus and incidence of cancer

Change in mucosal microbiome is associated with various types of cancer in digestive tract. We hypothesized that microbial communities in the esophageal endoscopic wash fluids reflects resident flora in esophageal mucosa that is associated with esophageal carcinoma (EC) risk and/or directly correlates microbiome derived from EC tumor tissue. Studying microbial communities in esophageal endoscopic wash samples would be therefore useful to predict the incidence or risk of EC. We examined microbial communities of the endoscopic wash samples from 45 primary EC and 20 respective non-EC controls using 16S rRNA V3-V4 amplicon sequencing. The result was also compared with microbial communities in matched endoscopic biopsies from EC and non-cancerous esophageal mucosa. Compared with non-EC controls, 6 discriminative bacterial genera were detected in EC patients. Among them, relative abundance ratio of Prevotella and Shuttlewarthia, as well as decrease of genus Prevotella presented good prognostic performance to discriminate EC from controls (area under curve, 0.86, 0.82, respectively). Multivariate analysis showed occurrence of EC was an independent factor associated with decrease of this bacteria. Abundance of genus Prevotella in the esophageal endoscopic wash samples was significantly correlated with the abundance of this bacteria in the matched endoscopic biopsies from non-cancerous esophageal mucosa but not in the EC tissues. Our findings suggest that microbiome composition in the esophageal endoscopic wash samples reflects resident flora in the esophagus and significantly correlates with the incidence of EC.

Pneumococcal carriage and disease in adults in England 2011-2019: the importance of adults as a reservoir for pneumococcus in communities.

Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood. Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years. In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children. The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.

The Acid Roles of PtSn@Al2O3 in the Synthesis and Performance of Propane Dehydrogenation.

In this study, a PtSn/Al2O3 catalyst with bimetallic uniform distribution in the sphere was synthesized. The PDH performance and characterization analyses, such as with FTIR, XPS, and NH3-TPD, were investigated. The effects of acid on the PDH performance were analyzed. Citric acid (CA) acted as a competing adsorbent in the preparation process of the PtSn/Al2O3 catalyst to synthesize the uniform catalyst. Water washing and alkali-treated samples were also studied. SEM line scanning revealed that increased the apparent concentration of Pt metal from 0.23 to 0.30 with citric acid. In contrast to the fresh PtSn/Al2O3 catalyst, the addition of citric acid increased the PDH selectivity from 74% to 93%. After alkali or water washing treatments, the catalyst's selectivity further increased to 96%. Strong acid sites promoted the breaking of C-C bonds during the PDH reaction, resulting in more methane and ethylene byproducts, and decreased catalyst selectivity for fresh PtSn/Al2O3. From the PDH reaction thermodynamic analysis, a relatively sub-atmospheric pressure environment with a lower propane pressure could be the reasonable choice.

Assessment of Microbial Contamination and Metabolite Exposure in Cosmetic Products Used in Women's Beauty Salons.

An important route of microbial transmission is the shared use of these products in beauty salons. We aimed to investigate level of contamination with microorganisms and their metabolites in shared-use cosmetics in several women's beauty salons. Bacterial and fungal strains from 320 opened/used cosmetic samples were identified according to the Iranian standards for microbial quality of cosmetic products in Golestan Province (North of Iran) during Jul-Sep 2021. In order to assess production of toxins and protease by the predominant bacterial and fungal isolates, multiplex-polymerase chain reaction and the Lowry protein assay were performed, respectively. Microbial contamination was detected in 180 samples (56.5%), and the highest and lowest rates of microbial contamination were related to skin products (63.88%) and eye beauty products (20%), respectively. The highest level of S. aureus contamination (> 4,000 colony-forming units/g) was found in toner and face wash samples, and the highest level of C. albicans contamination was seen in lipstick samples (>20,000 colony-forming units/g). Only one (2%) S. aureus isolate produced staphylococcal enterotoxin B, while 3 out of 12 (25%) C. albicans isolates were able to produce protease. The shared-used health and beauty products, face products, in the study area are heavily contaminated. Therefore, it is essential to store used cosmetics in dry and cool places, establish strict inspection regulations for cosmetic products before and after entering the market, and increase awareness of beauty salon workers regarding the appropriate use, sanitary control, and maintenance of health and beauty products.

Host obesity impacts genetic variation in influenza A viral populations.

Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Here, we investigated the impact of host obesity on influenza A virus (IAV) genetic variation using a diet-induced obesity ferret model and the A/Hong Kong/1073/1999 (H9N2) strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of mutations throughout the genome that were specific to obese hosts and that were preserved during transmission between hosts. Despite detection of obese-specific variants, the overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin and polymerase genes (PB2 and PB1). We also identified defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but the overall DVG diversity and dynamics did not differ between the two groups. Our study suggests that obesity may result in a unique selective environment impacting intrahost IAV evolution, highlighting the need for additional genetic and functional studies to confirm these effects.IMPORTANCEObesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative-sense single-stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

Viral load in hospitalized infants with respiratory syncytial virus bronchiolitis: a three-way comparative analysis.

Viral load measurement ofRespiratory syncytial virus (RSV) in acute bronchiolitis depends on specimen collection, viral load quantification, and transport media.The aim of this study wasto investigate viral load in three-way-comparative analyses; nasal swab versus nasal wash, quantitative real-time polymerase chain reaction (RT-PCR) versus cell tissue culture, and various transport media.A prospective cohort study of infants aged < 12 months, admitted to the Soroka Medical Center, due to acute bronchiolitis, was conducted. Two nasal swabs and two nasal wash samples (in UTM and VCM) were collected from each infant upon admission and after 48h. Samples were immediately stored at -80 °C and tested at Viroclinics DDL (Rotterdam, Netherlands). Quantitative RT-PCR and quantitative virus culture were performed using tissue culture infective dose (TCID50). Spearman's correlation coefficient test assessed the correlation between the different methods, viral load, and clinical severity score. One hundred samples were collected from 13 infants (mean age 5.7 ± 3.8 months, 46% males). Twelve patients were RSV-A positive, and one was RSV-B positive. A high correlation was found between transport media- UTM and VCM (0.92, P < 0.001) and between nasal swabs and nasal wash samples (0.62, P = 0.02). RSV signals were higher in nasal wash than in swabs. PCR signals were lower in the second collection compared to the first. No correlation was found between viral load and clinical severity. Conclusion: RSV viral load is comparable across nasal wash, nasal swabs, and various transport media. However, it did not correlate with clinical severity, probably due to the limited sample size. Broader analyses are warranted. What is Known: • Viral load measurement in Respiratory Syncytial Virus (RSV) bronchiolitis depends on specimen collection, viral load quantification, and transport media. • The COVID-19 pandemic underscored the paramount significance of proper specimen collection, notably through nasal swabs. What is New: • RSV viral load was investigated in three-way-comparative analyses. • RSV viral load correlated well across PCR and tissue culture, nasal wash and swabs, and various transport media.RSV viral load did not correlate with clinical severity.

Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.

Elevated neutrophils and reduced NK cells are associated with altered tear molecular signatures and clinical sequelae of chronic ocular Stevens-Johnson syndrome

PurposeStevens-Johnson syndrome (SJS) is characterised as an immuno-inflammatory condition with potentially blinding ocular sequelae. Therefore, we have investigated the ocular surface immune cell profile and correlated it with secreted tear molecular factors and clinical ocular sequelae in SJS patients. Methods21 patients (42 eyes) with chronic ocular SJS and 16 healthy controls (20 eyes) were included in the study. Severity, types of keratopathies and ocular surface (OS) manifestations were determined. OS wash samples from study subjects were used to determine the status of 13 immune cell subsets using flow cytometry. Levels of 42 secreted immuno-inflammatory factors were measured by flow cytometry-based multiplex ELISA in tear samples. ResultsNeutrophils (Total, activated), neutrophils/NK cells ratio, neutrophils/T cells ratio were significantly (p < 0.05) elevated in SJS, while, proportions of T cells and NKT cells were significantly lower in SJS patients. Positive association between neutrophils and chronic ocular surface complication score (COCS) was observed, whereas, a negative association was noted between NK cells and COCS. Tear fluid levels of IL-6, IL-8, IL-18, IFNα/β/γ, TNFα, LIF, IL-8, HGF, sTNFR-I, NGAL, Granzyme, Perforins, MMP9/TIMP1 ratio were significantly higher in SJS. Loss of Limbal niche correlated significantly with immune profile and clinical sequelae. Increased neutrophils, decreased NK cells and specific set of altered secreted immuno-inflammatory mediators including bFGF, and IL-8 were observed in SJS patients with different types of keratopathies compared to those without keratopathy. ConclusionDistinct ocular surface immune profile variations were observed to correlate with clinical stages of chronic ocular SJS. Our findings uncover novel mechanisms and potential for targeted therapy in chronic ocular SJS patients.

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 &gt;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.

Impact of Respiratory Bacterial Findings on Patients with Chronic Pulmonary Aspergillosis.

Distinct bacterial strains may affect the prognosis of patients with chronic respiratory diseases. However, little is known about the clinical significance of respiratory bacteria in patients with chronic pulmonary aspergillosis (CPA), a progressive and debilitating disease caused by Aspergillus spp. This study aimed to analyze data obtained from CPA patients and their sputum or bronchial washing samples and investigate the prevalence and composition of respiratory bacteria and clinical implications. Patients and Methods. We retrospectively reviewed the data of patients diagnosed with CPA between March 2019 and February 2023 in a tertiary referral hospital. We assessed the clinical characteristics and overall and pneumonia-specific survival rates of patients with CPA based on the presence of bacteria. Results and Conclusions. We included 142 patients with CPA. The most commonly identified bacteria were Klebsiella pneumoniae (22.5%), followed by Pseudomonas aeruginosa (21.8%) and Escherichia coli (4.2%). Patients with isolated bacteria had a higher prevalence of older age, female sex, diabetes, and a history of extrathoracic malignancy than those without isolated bacteria (P = 0.024, 0.013, 0.021, and 0.034, respectively). Furthermore, over a median follow-up of 11 (4-21) months, the pneumonia-specific mortality rate was 13.4% (19/142), which was higher in patients with isolated bacteria than in those without (P = 0.045, log-rank test). Particularly, patients with the presence of P. aeruginosa had a significantly higher mortality rate from pneumonia than those without the presence of P. aeruginosa (adjusted hazard ratio, 3.34; P = 0.015). In conclusion, CPA patients with isolated bacteria, especially P. aeruginosa, showed higher mortality rates due to pneumonia. Performing tests to identify bacteria in the lower respiratory tract of patients with CPA may be helpful in predicting future prognosis. Further studies are required to validate these findings in diverse ethnic groups.

Identification and validation of PCDHGA12 and PRRX1 methylation for detecting lung cancer in bronchial washing sample.

Bronchoscopy is a frequently used initial diagnostic procedure for patients with suspected lung cancer (LC). Cytological examinations of bronchial washing (BW) samples obtained during bronchoscopy often yield inconclusive results regarding LC diagnosis. The present study aimed to identify molecular biomarkers as a non-invasive method for LC diagnosis. Aberrant DNA methylation is used as a useful biomarker for LC. Therefore, microarray-based methylation profiling analyses on 13 patient-matched tumor tissues at stages I-III vs. non-tumor tissues were performed, and a group of highly differentially methylated genes was identified. A subsequent analysis using bisulfite-pyrosequencing with additional tissues and cell lines revealed six methylated genes [ADAM metallopeptidase with thrombospondin type 1 motif 20, forkhead box C2 (mesenchyme forkhead 1), NK2 transcription factor related, locus 5 (Drosophila), oligodendrocyte transcription factor 3, protocadherin γ subfamily A 12 (PCDHGA12) and paired related homeobox 1 (PRRX1)] associated with LC. Next, a highly sensitive and accurate detection method, linear target enrichment-quantitative methylation-specific PCR in a single closed tube, was applied for clinical validation using BW samples from patients with LC (n=68) and individuals with benign diseases (n=33). PCDHGA12 and PRRX1 methylation were identified as the best-performing biomarkers to detect LC. The two-marker combination showed a sensitivity of 82.4% and a specificity of 87.9%, with an area under the curve of 0.891. Notably, the sensitivity for small cell LC was 100%. The two-marker combination had a positive predictive value of 93.3% and a negative predictive value of 70.7%. The sensitivity was higher than that of cytology, which only had a sensitivity of 50%. The methylation status of the two-marker combination showed no association with sex, age or stage, but was associated with tumor location and histology. In conclusion, the present study showed that the regulatory regions of PCDHGA12 and PRRX1 are highly methylated in LC and can be used to detect LC in BW specimens as a diagnostic adjunct to cytology in clinical practice.

Liquid biopsy of wash samples obtained via endoscopic ultrasound-guided fine-needle biopsy: Comparison with liquid biopsy of plasma in pancreatic cancer.

Pancreatic cancer (PC) has a poor prognosis and limited treatment options. Liquid biopsy, which analyzes circulating tumor DNA (ctDNA) in blood, holds promise for precision medicine; however, low ctDNA detection rates pose challenges. This study aimed to investigate the utility of wash samples obtained via endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) as a liquid biopsy for PC. A total of 166 samples (42 formalin-fixed paraffin-embedded [FFPE] tissues, 80 wash samples, and 44 plasma samples) were collected from 48 patients with PC for genomic analysis. DNA was extracted and quantified, and 60 significantly mutated genes were sequenced. The genomic profiles of FFPE tissues, wash samples, and plasma samples were compared. Finally, the ability to detect druggable mutations in 80 wash samples and 44 plasma samples was investigated. The amount of DNA was significantly lower in plasma samples than in wash samples. Genomic analysis revealed a higher detection rate of oncogenic mutations in FFPE tissues (98%) and wash samples (96%) than in plasma samples (18%) and a comparable detection rate in FFPE tissues and wash samples. Tumor-derived oncogenic mutations were detected more frequently in wash samples than in plasma samples. Furthermore, the oncogenic mutations detection rate remained high in wash samples at all PC stages but low in plasma samples even at advanced PC stages. Using wash samples was more sensitive than plasma samples for identifying oncogenic and druggable mutations. The wash sample obtained via EUS-FNB is an ideal specimen for use as a liquid biopsy for PC.