Lavage Samples Research Articles

Clinical characteristics of patients with diffuse alveolar hemorrhage diagnosed by cytological examination of 1000 bronchoalveolar lavage samples.

Diffuse alveolar hemorrhage (DAH) is a life-threatening condition due to the extravasation of blood in the alveoli, resulting in hypoxemia and even acute respiratory distress syndrome. This study aimed to describe the clinico-radio-pathological profile of patients diagnosed with DAH and classify it into immune and nonimmune DAH. This was a retrospective analytical study. Of a total of 1000 cases of bronchoalveolar lavage fluids (BALF) received for cytological examination, patients fulfilling the clinical, radiological, and laboratory details of cases satisfying the clinical and cytological criteria of DAH (n=47) were studied. The most common cause of immune DAH was ANCA-associated vasculitis (n=13, 27.6%), and that of nonimmune DAH was infections (n=10, 21.3%). Twenty-nine patients (61.7%) had hemoptysis. The most common radiological finding was ground-glass opacities (n=33, 70.2%). In univariate analysis, female sex, mean hemoglobin at admission, total leucocyte count (TLC), platelet count, and erythrocyte sedimentation rate (ESR) were significantly associated with immune-DAH. However, in multivariate analysis, female sex, higher TLC, high platelets, and high ESR were significantly associated with immune DAH. Patients were treated with corticosteroids (n=25, 46.3%), intravenous cyclophosphamide (n=12, 22.2%), plasma exchange (n=7, 13.0%), intravenous immunoglobulin (n=5, 9.3%) and rituximab (n=5, 9.3%). The overall mortality was 8.5% (n=4). DAH is a life-threatening syndrome that may be classified into immune and nonimmune DAH. Immune-DAH requires aggressive management, whereas nonimmune DAH cases respond best to conservative management.

Loop-mediated Isothermal Amplification Test for Rapid Identification of Clinical Nocardia Isolates

Background: Nocardia is a Gram-positive and partially acid-fast bacterium. The species are widely distributed in the environment and cause severe human infections. Nocardiosis is not easily identifiable due to the lack of pathognomonic clinical signs. Objectives: The present study was designed to develop and evaluate a simple and quick method based on a loop-mediated isothermal amplification (LAMP) assay for detecting Nocardia spp isolated from bronchoalveolar lavage (BAL) samples. Methods: In this cross-sectional study, 357 BAL samples were collected from two teaching hospitals. The polymerase chain reaction (PCR) was performed using a set of species-specific primers for the 16S rRNA gene. Kinyoun acid-fast staining and culture were done on the Sabouraud dextrose plate. The optimal LAMP reaction condition was set at 65°C for 45 min, with the recognition limit as 1 pg DNA/tube and 100 CFU/reaction. In addition to calcein and manganous ions, agarose gel electrophoresis was used to visualize the amplified LAMP products. Results: Out of 357 BAL samples, 0 (0.0%), 4 (1.1%), 9 (2.5%), and 10 (2.8%) Nocardia strains were identified by direct staining of partial acid-fast, streak culture plate, PCR, and LAMP methods, respectively. Conclusions: We developed a new LAMP technique for the recognition of Nocardia, which is fast, very precise, simple, and low-cost. According to our knowledge, this is the first report of the LAMP method to detect Nocardia in clinical samples.

The Cystic Fibrosis Upper and Lower Airway Metagenome.

The microbial metagenome in cystic fibrosis (CF) airways was investigated by whole-genome shotgun sequencing of total DNA isolated from nasal lavage samples, oropharyngeal swabs, and induced sputum samples collected from 65 individuals with CF aged 7 to 50 years. Each patient harbored a personalized microbial metagenome unique in microbial load and composition, the exception being monocultures of the most common CF pathogens Staphylococcus aureus and Pseudomonas aeruginosa from patients with advanced lung disease. The sampling of the upper airways by nasal lavage uncovered the fungus Malassezia restricta and the bacterium Staphylococcus epidermidis as prominent species. Healthy and CF donors harbored qualitatively and quantitatively different spectra of commensal bacteria in their sputa, even in the absence of any typical CF pathogen. If P. aeruginosa, S. aureus, or Stenotrophomonas maltophilia belonged to the trio of the most abundant species in the CF sputum metagenome, common inhabitants of the respiratory tract of healthy subjects, i.e., Eubacterium sulci, Fusobacterium periodonticum, and Neisseria subflava, were present only in low numbers or not detectable. Random forest analysis identified the numerical ecological parameters of the bacterial community, such as Shannon and Simpson diversity, as the key parameters that globally distinguish sputum samples from CF and healthy donors. IMPORTANCE Cystic fibrosis (CF) is the most common life-limiting monogenetic disease in European populations and is caused by mutations in the CFTR gene. Chronic airway infections with opportunistic pathogens are the major morbidity that determines prognosis and quality of life in most people with CF. We examined the composition of the microbial communities of the oral cavity and upper and lower airways in CF patients across all age groups. From early on, the spectrum of commensals is different in health and CF. Later on, when the common CF pathogens take up residence in the lungs, we observed differential modes of depletion of the commensal microbiota in the presence of S. aureus, P. aeruginosa, S. maltophilia, or combinations thereof. It remains to be seen whether the implementation of lifelong CFTR (cystic fibrosis transmembrane conductance regulator) modulation will change the temporal evolution of the CF airway metagenome.

Pulmonary Aspergillosis in Critically Ill COVID-19 Patients Admitted to the Intensive Care Unit: A Retrospective Cohort Study.

COVID-19-associated pulmonary aspergillosis (CAPA) is a life-threatening fungal infection that mainly affects critically ill patients. The aim of this study was to assess the incidence and clinical outcomes of putative CAPA in critically ill COVID-19 patients. This retrospective observational cohort study included 181 cases from 5 ICUs at Vienna General Hospital between January 2020 and April 2022. Patients were diagnosed with putative CAPA according to the AspICU classification, which included a positive Aspergillus culture in a bronchoalveolar lavage sample, compatible signs and symptoms, and abnormal medical imaging. The primary outcome was adjusted 60-day all-cause mortality from ICU admission in patients with vs. without putative CAPA. Secondary outcomes included time from ICU admission to CAPA diagnosis and pathogen prevalence and distribution. Putative CAPA was identified in 35 (19.3%) of 181 COVID-19 patients. The mean time to diagnosis was 9 days. Death at 60 days occurred in 18 of 35 (51.4%) patients with CAPA and in 43 of 146 (29.5%) patients without CAPA (adjusted HR (95%CI) = 2.15 (1.20-3.86, p = 0.002). The most frequently isolated Aspergillus species was Aspergillus fumigatus. The prevalence of putative pulmonary aspergillosis in critically ill COVID-19 patients was high and was associated with significantly higher mortality.

From Gut to Blood: Spatial and Temporal Pathobiome Dynamics during Acute Abdominal Murine Sepsis.

Abdominal sepsis triggers the transition of microorganisms from the gut to the peritoneum and bloodstream. Unfortunately, there is a limitation of methods and biomarkers to reliably study the emergence of pathobiomes and to monitor their respective dynamics. Three-month-old CD-1 female mice underwent cecal ligation and puncture (CLP) to induce abdominal sepsis. Serial and terminal endpoint specimens were collected for fecal, peritoneal lavage, and blood samples within 72 h. Microbial species compositions were determined by NGS of (cell-free) DNA and confirmed by microbiological cultivation. As a result, CLP induced rapid and early changes of gut microbial communities, with a transition of pathogenic species into the peritoneum and blood detected at 24 h post-CLP. NGS was able to identify pathogenic species in a time course-dependent manner in individual mice using cfDNA from as few as 30 microliters of blood. Absolute levels of cfDNA from pathogens changed rapidly during acute sepsis, demonstrating its short half-life. Pathogenic species and genera in CLP mice significantly overlapped with pathobiomes from septic patients. The study demonstrated that pathobiomes serve as reservoirs following CLP for the transition of pathogens into the bloodstream. Due to its short half-life, cfDNA can serve as a precise biomarker for pathogen identification in blood.

Tuberculosis with isolated atelectasis: a case report

Introduction Tuberculosis is a significant health issue in developing countries, with the World Health Organization reporting over 10 million cases worldwide in 2014. While methods exist for rapid investigation of Mycobacterium tuberculosis, radiological imaging may lead to an earlier suspicion, such as presence of cavitation. In this case report, a patient with only radiological finding being atelectasis will be presented. Case Presentation An 18 years old female patient with no specific medical background history had been evaluated at an outpatient clinic with complaints of cough and exertional dyspnea. After a fluoroquinolone treatment for 7 days given for a pericardiac density in chest radiography, patient was admitted due to limited clinical response. A chest tomography was performed while the patient was under wide spectrum antibiotics and atelectasis in right lower lobe was observed. Repeated Acid-Fast Bacilli smears and sputum samples were found negative and bronchoscopy sampling did not show any findings of obstruction. Mycobacterium tuberculosis complex DNA was later detected in the lavage samples for which antituberculosis treatment was initiated with a regimen of isoniazid 5 mg/kg, pyrazinamide 25 mg/kg, ethambutol 15 mg/kg, and rifampin 10mg/kg. Discussion The diagnosis of tuberculosis, in this case, was probably masked during the AFB smear testing due to a treatment regimen of fluoroquinolone. The general radiological definition of tuberculosis primarily consists of older studies, with the expected presentation being a combination of cavitation and infiltration. Normal lung imaging results had also been described for tuberculosis regarding chest radiography, and studies have reported the presence of atelectasis in patients with tuberculosis, as seen in tomography case series; however, the isolation of atelectasis had not been specified in the studies. Conclusion Pulmonary tuberculosis may present itself with a myriad of radiological findings. This presentation often complicates the diagnostic process; however, differential diagnosis can be achieved via a combination of clinical symptoms and rapid culture sampling.

Convergent dysbiosis of upper aerodigestive microbiota between patients with esophageal and oral cavity squamous cell carcinoma.

The bidirectional association between primary esophageal squamous cell carcinoma (ESCC) and oral cavity squamous cell carcinoma (OSCC) suggests common risk factors and oncogenic molecular processes but it is unclear whether these two cancers display similar patterns of dysbiosis in their upper aerodigestive microbiota (UADM). We conducted a case-control study to characterize the microbial communities in esophageal lavage samples from 49 ESCC patients and oral rinse samples from 91 OSCC patients using 16S rRNA V3-V4 amplicon sequencing. Compared with their respective non-SCC controls from the same anatomical sites, 32 and 45 discriminative bacterial genera were detected in ESCC and OSCC patients, respectively. Interestingly, 20 of them were commonly enriched or depleted in both types of cancer, suggesting a convergent niche adaptation of upper aerodigestive SCC-associated bacteria that may play important roles in the pathogenesis of malignancies. Notably, Fusobacterium, Selenomonas, Peptoanaerobacter and Peptostreptococcus were enriched in both ESCC and OSCC, whereas Streptococcus and Granulicatelia were commonly depleted. We further identified Fusobacterium nucleatum as the most abundant species enriched in the upper aerodigestive SCC microenvironment, and the higher relative abundances of Selenomonas danae and Treponema maroon were positively correlated with smoking. In addition, predicted functional analysis revealed several depleted (eg, lipoic acid and pyruvate metabolism) and enriched (eg, RNA polymerase and nucleotide excision repair) pathways common to both cancers. Our findings reveal a convergent dysbiosis in the UADM between patients with ESCC and OSCC, suggesting a shared niche adaptation of host-microbiota interactions in the pathogenesis of upper aerodigestive tract malignancies.

A pilot study exploring the association of bronchial bacterial microbiota and recurrent wheezing in infants with atopy.

Differences in bronchial microbiota composition have been found to be associated with asthma; however, it is still unclear whether these findings can be applied to recurrent wheezing in infants especially with aeroallergen sensitization. To determine the pathogenesis of atopic wheezing in infants and to identify diagnostic biomarkers, we analyzed the bronchial bacterial microbiota of infants with recurrent wheezing and with or without atopic diseases using a systems biology approach. Bacterial communities in bronchoalveolar lavage samples from 15 atopic wheezing infants, 15 non-atopic wheezing infants, and 18 foreign body aspiration control infants were characterized using 16S rRNA gene sequencing. The bacterial composition and community-level functions inferred from between-group differences from sequence profiles were analyzed. Both α- and β-diversity differed significantly between the groups. Compared to non-atopic wheezing infants, atopic wheezing infants showed a significantly higher abundance in two phyla (Deinococcota and unidentified bacteria) and one genus (Haemophilus) and a significantly lower abundance in one phylum (Actinobacteria). The random forest predictive model of 10 genera based on OTU-based features suggested that airway microbiota has diagnostic value for distinguishing atopic wheezing infants from non-atopic wheezing infants. PICRUSt2 based on KEGG hierarchy (level 3) revealed that atopic wheezing-associated differences in predicted bacterial functions included cytoskeleton proteins, glutamatergic synapses, and porphyrin and chlorophyll metabolism pathways. The differential candidate biomarkers identified by microbiome analysis in our work may have reference value for the diagnosis of wheezing in infants with atopy. To confirm that, airway microbiome combined with metabolomics analysis should be further investigated in the future.

Exosomal hsa-let-7g-3p and hsa-miR-10395-3p derived from peritoneal lavage predict peritoneal metastasis and the efficacy of neoadjuvant intraperitoneal and systemic chemotherapy in patients with gastric cancer.

The prognosis of advanced gastric cancer (GC) invading the gastric serosa remains poor, mainly owing to high incidence of peritoneal recurrence. Patients with peritoneal metastases are often treated with neoadjuvant intraperitoneal and systemic chemotherapies (NIPS). Good responders to NIPS often undergo conversion gastrectomy. This study aims to explore biomarkers predicting the occurrence of peritoneal metastasis (PM) and evaluating the efficacy of NIPS in GC patients. We collected six peritoneal lavage (PL) samples from two patients with PM, two without PM, and two with diminished PM after NIPS via intraperitoneal access ports. We equally isolated microRNAs from exosomes derived from PL samples for deep sequencing. Two microRNAs (hsa-let-7g-3p and hsa-miR-10395-3p) were identified, and their expression levels were examined in PL samples of 99 GC patients using qRT-PCR. Moreover, we performed in vivo and in vitro functional assays to investigate effects of these microRNAs on metastasis and chemoresistance of GC cells. Exosomal microRNA expression profiling of six PL samples indicated that the microRNA signature in exosomes of PLs from patients with diminished PM was similar to that from patients without PM. Expression levels of hsa-let-7g-3p and hsa-miR-10395-3p were associated with PM. In vivo and in vitro functional assays confirmed that hsa-let-7g-3p and hsa-miR-10395-3p are involved in GC metastasis and chemoresistance. PL-derived exosomes in GC contain large amounts of microRNAs related to PM. Moreover, hsa-let-7g-3p and hsa-miR-10395-3p could be used as biomarkers predicting PM and NIPS efficacy and are involved in GC metastasis and chemoresistance.

Detection of Porcine Deltacoronavirus RNA in the Upper and Lower Respiratory Tract and Biliary Fluid and the Effect of Infection on Serum Cholesterol Levels and Blood T Cell Population Frequencies in Gnotobiotic Piglets

Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further characterize PDCoV infection by investigating the presence of virus in respiratory and biliary tissues or fluids; T cell population frequencies in blood; and altered serum cholesterol levels. Twelve, 6-day-old, gnotobiotic piglets were inoculated oronasally with PDCoV OH-FD22 (2.6 × 107 FFU/pig). Six control piglets were not inoculated. Rectal swab (RS), nasal swab (NS), nasal wash (NW), bronchoalveolar lavage (BAL), and biliary fluid (BF) samples were collected at 2, 4, and 7 days post-inoculation (DPI) and tested for PDCoV RNA by RT-qPCR. Blood T cell populations and serum cholesterol levels were determined by flow cytometry and a colorimetric assay, respectively. Moderate to high, and low to moderate titers of PDCoV RNA were detected in RS and in NS, NW, BAL, and BF samples, respectively, of inoculated piglets. There were trends toward decreased CD4+CD8-, CD4-CD8+, and CD4+CD8+ blood T cell frequencies in inoculated piglets. Furthermore, serum cholesterol levels were increased in inoculated piglets. Overall, we found that PDCoV infection does not exclusively involve the intestine, since the respiratory and biliary systems and cholesterol metabolism also can be affected.

Further evidence of a type 2 inflammatory signature in chronic obstructive pulmonary disease or emphysema

BackgroundThere is increasing recognition of a type 2 (T2) inflammatory pattern in a subset of patients with chronic obstructive pulmonary disease (COPD) or emphysema, characterized by blood and airway eosinophilia. The mechanism underlying this is not well established. The recognition that CD125 (interleukin [IL]-5 receptor alpha) is expressed on some lung neutrophils and eosinophils in patients with asthma led us to speculate that CD125 may also be expressed on lung neutrophils in patients with COPD or emphysema. ObjectiveTo interrogate the expression of CD125 on lung neutrophils (and, when present, eosinophils) in patients with COPD/emphysema and identify a meaningful biomarker to predict neutrophil CD125 expression, including other markers of T2 inflammation. MethodsWe obtained blood and bronchoalveolar lavage (BAL) samples from patients with physician-diagnosed COPD/emphysema undergoing a clinically indicated bronchoscopy. ResultsWe found that a highly variable percentage of BAL neutrophils indeed expressed surface CD125 (0%-78.7%), with obvious clustering of CD125high and CD125low patterns. No correlation was found with clinical characteristics, blood or BAL eosinophil or neutrophil counts, BAL cytokines, or BAL eosinophil CD125 expression. ConclusionWe conclude that, similar to asthma, lung neutrophils from patients with COPD display interleukin-5 receptor alpha (CD125) on their surface. This along with the frequent presence of IL-4 and IL-5 in airway fluid further suggests a possible role of the T2 pathway in contributing to COPD severity. Trial RegistrationClinicalTrials.gov Identifier: NCT03984799

Hemosiderin-laden macrophages in bronchoalveolar lavage samples of children with bronchopulmonary dysplasia.

To evaluate the prevalence of hemosiderin-laden macrophages in children with bronchopulmonary dysplasia (BPD) and assess for an association between hemosiderin-laden macrophages and pulmonary arterial hypertension. Retrospective case-control study of infants and children with and without BPD who underwent bronchoscopy with bronchoalveolar lavage (BAL) the at Children's Hospital of Philadelphia between 2012 and2021. BAL from 205 children with BPD and 106 controls without BPD matched for tracheostomy, infection, and age were reviewed for hemosiderin-laden macrophages. Seventy-one individuals (34.6%) with BPD had a BAL with 10% or more hemosiderin-laden macrophages compared with 3 (2.8%) controls (P<.0001; OR, 18.19; 95% CI, 5.57-59.41). Patients with pulmonary hypertension by echocardiogram (P=.04; OR, 3.69; 95% CI, 1.05-12.96) or an elevated mean pulmonary artery pressure during cardiac catheterization, rs (14)=0.56, P=.04, were more likely to have elevated hemosiderin-laden macrophages on BAL samples less than 60days from bronchoscopy. After adjusting for birth weight, gestational age, BPD grade, and age at the time of bronchoscopy using logistic regression, pulmonary hypertension was associated with a higher odds of hemosiderin-laden macrophages of 10% or more (P=.02; OR, 6.37; 95% CI, 1.28-31.87). No association was observed between hemosiderin-laden macrophages and sex, race, gestational age, birth weight, tracheostomy, or infectious studies. This retrospective study revealed increased hemosiderin-laden macrophages in BAL samples from patients with BPD and a significant association with pulmonary arterial hypertension. It is unclear whether elevated hemosiderin-laden macrophages within BPD contributes to the pathogenesis of lung and pulmonary vascular disease or is simply a biomarker of pulmonary arterial hypertension.

Effects of a water-soluble formulation of tylvalosin on disease caused by porcine reproductive and respiratory syndrome virus alone in sows or in combination with Mycoplasma hyopneumoniae in piglets

BackgroundThe effect of a water-soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) on disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (Mhyop) was investigated in two animal studies. In a PRRSV challenge model in pregnant sows (n = 18), six sows received water medicated at target dose of 5 mg tylvalosin/kg body weight/day from 3 days prior to challenge until the end of gestation. Six sows were left untreated, with a third group remaining untreated and unchallenged. Sows were challenged with PRRSV-2 at approximately 85 days of gestation. Cytokines, viremia, viral shedding, sow reproductive parameters and piglet performance to weaning were evaluated. In a dual infection study (n = 16), piglets were challenged with Mhyop on days 0, 1 and 2, and with PRRSV-1 on day 14 and euthanized on day 24. From day 10 to 20, eight piglets received water medicated at target dose of 20 mg tylvalosin/kg body weight/day and eight piglets were left untreated. Cytokines, viremia, bacteriology and lung lesions were evaluated.ResultsIn the PRRSV challenge study in pregnant sows, tylvalosin significantly reduced the levels of serum IL-8 (P < 0.001), IL-12 (P = 0.032), TNFα (P < 0.001) and GM-CSF (P = 0.001). IL-8 (P = 0.100) tended to be lower in uterus of tylvalosin sows. All piglets from tylvalosin sows surviving to weaning were PRRSV negative in faecal swabs at weaning compared to 33.3% PRRSV positive piglets from untreated sows (P = 0.08).In the dual challenge study in piglet, tylvalosin reduced serum IL1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-1α, IL-13, IL-17A, IL-18, GM-CSF, TGFβ1, TNFα, CCL3L1, MIG, PEPCAM-1 (P < 0.001) and increased serum IFNα, IL-1ra and MIP-1b (P < 0.001). In the lungs, tylvalosin reduced IL-8, IL-10 and IL-12 compared to untreated pigs (P < 0.001) and tended to reduce TNFα (P = 0.082). Lung lavage samples from all tylvalosin treated piglets were negative for Mhyop (0 cfu/mL) compared to the untreated piglets which had mean Mhyop counts of 2.68 × 104 cfu/mL (P = 0.023).ConclusionOverall, tylvalosin reduced both local and systemic proinflammatory cytokines after challenge with respiratory pathogens in sows and in piglets. Tylvalosin was effective in reducing Mhyop recovery from the lungs and may reduce virus shedding in piglets following transplacental PRRSV infection in sows.

Seroprevalence of Tick-Borne Encephalitis (TBE) Virus Antibodies in Wild Rodents from Two Natural TBE Foci in Bavaria, Germany

Tick-borne encephalitis (TBE) is Eurasia's most important tick-borne viral disease. Rodents play an important role as natural hosts. Longitudinal studies on the dynamics of the seroprevalence rates in wild rodents in natural foci over the year are rare, and the dynamics of the transmission cycle still need to be understood. To better understand the infection dynamics, rodents were captured in a capture-mark-release-recapture-study in two natural foci in Bavaria, Germany, monthly from March 2019 to October 2022. Overall, 651 blood and thoracic lavage samples from 478 different wild rodents (Clethrionomys glareolus and Apodemus flavicollis) were analyzed for antibodies against tick-borne encephalitis virus (TBEV) by indirect immunofluorescence assay (IIFA) and confirmed using a serum neutralization test (SNT). Furthermore, a generalized linear mixed model (GLMM) analysis was performed to investigate ecological and individual factors for the probability of infection in rodents. Clethrionomys glareolus (19.4%) had a higher seroprevalence than A. flavicollis (10.5%). Within Cl. glareolus, more males (40.4%) than females (15.6%) were affected, and more adults (25.4%) than juveniles (9.8%). The probability of infection of rodents rather depends on factors such as species, sex, and age than on the study site of a natural focus, year, and season. The high incidence rates of rodents, particularly male adult bank voles, highlight their critical role in the transmission cycle of TBEV in a natural focus and demonstrate that serologically positive rodents can be reliably detected in a natural focus regardless of season or year. In addition, these data contribute to a better understanding of the TBEV cycle and thus could improve preventive strategies for human infections.

O03 Rapid Infection Diagnostics in Cirrhosis (RIDiC): a novel molecular-based approach to address the ever increasing challenge of antimicrobial resistant infections in decompensated cirrhosis and acute-on-chronic liver failure

Abstract Background Global prevalence of MDR infections in cirrhotic patients has risen to 34% and associates with worsening clinical trajectory and mortality (Fernandez J et al. 2019). A major unmet need in addressing the challenge of antimicrobial resistance (AMR) in cirrhosis is rapid pathogen diagnosis and antimicrobial resistance (AMR) (Patel VC et al. 2019). These can facilitate selection of more effective antibiotic therapy, reducing selection for MDR organisms. The Curetis Unyvero™ platform (CUp) employs multiplex PCR technology for pathogen and AMR gene detection from multiple bodily tissues and fluids requiring minimal pre- processing. The investigator-initiated pilot study aimed to assess the utility of this platform for hospitalized cirrhotic patients. Methods Ascitic fluid, bronchoalveolar lavage (BAL) and blood culture samples were obtained from hospitalized cirrhotic patients with acute decompensation (AD) or acute-on-chronic liver failure (ACLF), in whom there was clinical suspicion of infection. Samples were tested using the IAI (intra-abdominal infection) panel, HPN (hospital pneumonia) panel and BCU (blood culture) panel based on sample type available. Results were not shared with clinicians for decision-making. Data were compared with standard microbiology diagnostics. Results 31 samples from 21 patients were tested: ascites (25), BAL (4), blood culture (2). Time to positive culture result of all samples was reduced with CUp compared with standard microbiology (4.59 versus 73.5 h). CUp showed 100% specificity (12/12) and detected the same organism in 100% of cases (10/12). In 6 samples, CUp identified pathogens which were consistent with clinical evidence of spontaneous bacterial peritonitis and previous positive isolates which standard microbiological diagnostics failed to identify. AMR genes identified by the CUp correlated with AMR patterns reported by standard diagnostics in 100% (5/5) of cases. In 2 patients, broad-spectrum antibiotic therapy continued although the CUp supported infection resolution. Four patients in whom CUp screening was negative continued to receive antibiotics whilst awaiting negative standard cultures that arrived 72 h later. CUp did not identify an Aspergillus species in one BAL sample as this is not part of the BAL panel. Conclusions These preliminary data suggest CUp results are compatible with standard laboratory diagnostics. The CUp may be utilized for earlier infection diagnosis and improve the targeting and de-escalation of antimicrobial therapy in AD and ACLF. These are crucial components in enhancing antibiotic stewardship strategies and to reduce MDR infections (Figure 1). Further work is however required to evaluate cost implications, validation and impact on clinical outcomes and mortality in cirrhosis.

Emergence of Raoultella ornithinolytica Producing Beta Lactamase Enzyme in Different Clinical Specimens in Erbil City

Background: Raoultella ornithinolytica should never be regarded as merely a saprophytic bacterium that occasionally contaminates bronchial lavage or other deep respiratory samples or surgical sites is an underreported, emerging hospital-acquired infection and is particularly related with invasive operations.R. ornithinolytica isolates have significant antimicrobial resistance rates, and physicians should be aware of this so that, before accurate microbiological data are received, an immediate broad-spectrum antibiotic treatment can be instituted..&#x0D; Objectives: The goal of our study was to retrospectively examine R. ornithinolytica, which was isolated from a variety of clinical samples in Erbil, and to examine its epidemiology, antibiotic sensitivity patterns, and ability to produce the enzyme extend spectrum beta lactamase.&#x0D; Methods: A total of 2350 samples were collected from three different sources(Urine ,High vaginal swab and wound), collected from female and male from patient attending Rizgary hospital and Central lab in Erbil city during the July 2017 to Februry 2018 . Only 10 cases had been identified as R. ornithinolytica and all isolates isolated and identified by using microscopical, morphological , biochemical tests and Vitek 2 compact system . Also antibiotics susceptibility testing was performed by using Vitec 2 compact system according to the standard protocol against 13 antibiotics which are (Ampicillin /Sulbactam , Piperacillin/ Tazobactam, Cefazolin, Ceftazidim , Ceftriaxon , Cefepime, Levofloxacin , Trimethoprim/ Sulfamethoxazol, Gentamycin, Tobramycin, Ertapenem, Imipenem, Ciprofloxacin).&#x0D; Results: Only ten R. ornithinolytica isolates isolated from 2350 distribution according to their source of isolation High vaginal swab 6(0.23%) appeared to be the most dominant specimen than other specimens followed by urine sample 3(0.13%) then wound 1(0.04%).The highest percentage of R. ornithinolytica. isolated from female .Most isolates from high vaginal swab sample 6(0.26%) followed by urine 3 (0.13%) then wound 1(0.04), when performing of antibiotic susceptibility the highest resistances rate were to Cefazolin and Trimethoprim / Sulfamethoxazol 10 (100%) followed by Ampicillin / Sulbactam , Piperacillin / Tazobactam, Ceftriaxon , Gentamycin , Tobramycin and Ciprofloxacin 7(70%) for each, 6(60%)isolates ESBL producer, all isolated R. ornithinolytica were resistance to more than 8 antibiotics and two isolates completely resistance to all thirteen antibiotics.&#x0D; Conclusion: The overall prevalence of R. ornithinolytica isolates was low in Erbil city and we can be observed and isolated from varies clinical samples and causes serious infections and susceptibility to some antibiotics are low and also most isolates multiresistance and ESBL producers and These findings offer a reliable measure of the prevalence of Raoultella ornithinolytica in our region and provide a baseline for future studies that will enable the monitoring of trends over time. If current resistance trends continue, high societal and economic costs can be anticipated; better management of infections caused by resistanceR.ornithinolytica is becoming essential.

Specific Parameters of Detection of Fungi of the Genus &lt;i&gt;Aspergillus&lt;/i&gt; in Respiratory Organs of Pulmonary Tuberculosis Patients

The objective: to study specific parameters of detection of fungi of the genus Aspergillus in respiratory organs of pulmonary tuberculosis patients.Subjects and Methods. In 2019-2021, the samples of sputum or bronchoalveolar lavage of 520 patients above 18 years old with confirmed pulmonary tuberculosis and HIV-negative status were tested for molds. Also, all patients had their sputum or BAL tested for acid fast mycobacteria (AFB), cultures of M. tuberculosis (MBT), and DNA of tuberculous mycobacteria.Results. Positive culture results for fungi were obtained in 50/520 (9.62%; 95% CI 7.37-12.45, Wilson test) pulmonary tuberculosis patients. Species identification revealed fungi of the genus Aspergillus in 46/50 (92%) of them.In 37/46 (80.43%; 95% CI 66.83-89.35) patients, a single type (one species of Aspergillus) was identified, in 9/46 (19.57%; 95% CI 10.65-33.17) two types were identified. A. fumigatus (16/37 isolates, 43.24%) and A. niger (13/37 isolates, 35.14%) prevailed among single types of cultures. A. niger + A. flavus (3/9 isolates, 33.33%) was the most frequent combination. The ratio of the effectiveness of methods for detecting fungi such as culture/direct microscopy/galactomannan antigen in serum and BAL was 100/84.78/4.35% and 67.39%, respectively. Thus, BAL is more preferable than serum for detection of galactomannan antigen in patients with tuberculosis.Only in 8/46 (17.4%) patients, bacterial excretion was detected by culture, which is typical for the active phase of the course of tuberculosis. In the remaining 38 (82.6% 95% CI 69.28-90.91) patients, no tuberculous mycobacteria were detected by culture, which was typical of stabilization and cure of tuberculosis.

Predictive markers related to local and systemic inflammation in severe COVID-19-associated ARDS: a prospective single-center analysis

BackgroundAs the COVID-19 pandemic strains healthcare systems worldwide, finding predictive markers of severe courses remains urgent. Most research so far was limited to selective questions hindering general assumptions for short- and long-term outcome.MethodsIn this prospective single-center biomarker study, 47 blood- and 21 bronchoalveolar lavage (BAL) samples were collected from 47 COVID-19 intensive care unit (ICU) patients upon admission. Expression of inflammatory markers toll-like receptor 3 (TLR3), heme oxygenase-1 (HO-1), interleukin (IL)-6, IL-8, leukocyte counts, procalcitonin (PCT) and carboxyhemoglobin (CO-Hb) was compared to clinical course. Clinical assessment comprised acute local organ damage, acute systemic damage, mortality and outcome after 6 months.ResultsPCT correlated with acute systemic damage and was the best predictor for quality of life (QoL) after 6 months (r = − 0.4647, p = 0.0338). Systemic TLR3 negatively correlated with impaired lung function (ECMO/ECLS: r = − 0.3810, p = 0.0107) and neurological short- (RASS mean: r = 0.4474, p = 0.0023) and long-term outcome (mRS after 6 m: r = − 0.3184, p = 0.0352). Systemic IL-8 correlated with impaired lung function (ECMO/ECLS: r = 0.3784, p = 0.0161) and neurological involvement (RASS mean: r = − 0.5132, p = 0.0007). IL-6 in BAL correlated better to the clinical course than systemic IL-6. Using three multivariate regression models, we describe prediction models for local and systemic damage as well as QoL. CO-Hb mean and max were associated with higher mortality.ConclusionsOur predictive models using the combination of Charlson Comorbidity Index, sex, procalcitonin, systemic TLR3 expression and IL-6 and IL-8 in BAL were able to describe a broad range of clinically relevant outcomes in patients with severe COVID-19-associated ARDS. Using these models might proof useful in risk stratification and predicting disease course in the future.Trial registration The trial was registered with the German Clinical Trials Register (Trial-ID DRKS00021522, registered 22/04/2020).

The rapid detection of respiratory pathogens in critically ill children

PurposeRespiratory infections are the most common reason for admission to paediatric intensive care units (PICU). Most patients with lower respiratory tract infection (LRTI) receive broad-spectrum antimicrobials, despite low rates of bacterial culture confirmation. Here, we evaluated a molecular diagnostic test for LRTI to inform the better use of antimicrobials.MethodsThe Rapid Assay for Sick Children with Acute Lung infection Study was a single-centre, prospective, observational cohort study of mechanically ventilated children (> 37/40 weeks corrected gestation to 18 years) with suspected community acquired or ventilator-associated LRTI. We evaluated the use of a 52-pathogen custom TaqMan Array Card (TAC) to identify pathogens in non-bronchoscopic bronchoalveolar lavage (mini-BAL) samples. TAC results were compared to routine microbiology testing. Primary study outcomes were sensitivity and specificity of TAC, and time to result.ResultsWe enrolled 100 patients, all of whom were tested with TAC and 91 of whom had matching culture samples. TAC had a sensitivity of 89.5% (95% confidence interval (CI95) 66.9–98.7) and specificity of 97.9% (CI95 97.2–98.5) compared to routine bacterial and fungal culture. TAC took a median 25.8 h (IQR 9.1–29.8 h) from sample collection to result. Culture was significantly slower: median 110.4 h (IQR 85.2–141.6 h) for a positive result and median 69.4 h (IQR 52.8–78.6) for a negative result.ConclusionsTAC is a reliable and rapid adjunct diagnostic approach for LRTI in critically ill children, with the potential to aid early rationalisation of antimicrobial therapy.

Assessment of Epithelial Lining Fluid Partitioning of Systemically Administered Monoclonal Antibodies in Rats

For systemically administered monoclonal antibodies (mAbs) with pharmacological targets in the epithelial lining fluid (ELF), information on the partitioning of mAb between plasma and ELF is instrumental for dose predictions. Bronchoalveolar lavage (BAL) combined with measurements of urea as indicator of sample dilution is often used to estimate ELF concentrations of a drug. However, unbalanced extraction of mAb and urea could potentially lead to a systematic bias in the back-calculated ELF concentration. In the present study 0.5, 1, or 4 mL phosphate-buffered saline was instilled to lungs of rats to obtain lavage samples after systemic dosing of mAb and tool small molecule (n≥4/group). Furthermore, extraction of urea, mAb and the small molecule was assessed by repeatedly lavaging the lung (n = 4). There was no statistically significant difference in the calculated partitioning into ELF between the evaluated instillation volumes. Repeated BAL demonstrated that urea and the small molecule were extracted from other sources than the ELF. In contrast, there was limited to none in-flow of mAb into the lavage fluid. The unbalanced extraction of urea and mAb could theoretically result in underestimated ELF concentrations and the calculated partitioning of 0.17±0.062 might therefore constitute a lower boundary for the true partitioning.