Lavage Samples Research Articles

Zastupljenost respiratornih virusa u infekcijama donjih partija respiratornog trakta u dečjem uzrastu

Introduction: Acute viral infections of the lower respiratory tract are of great importance for mortality in children worldwide. The most common causative agents include human respiratory syncytial virus (HRSV), human metapneumovirus, parainfluenza virus, influenza virus and adenoviruses. The application of molecular methods has significantly contributed to the identification and determination of the frequency of respiratory viruses in these infections. Aim: The aim of this study was to determine the frequency of different viruses that cause lower respiratory tract infections in the pediatric population using panel PCR test from bronchoalveolar lavage samples. Material and methods: Eighteen samples of bronchoalveolar lavage of children younger than 5 years with a diagnosis of pneumonia and antigenic confirmation of the presence of HRSV were analyzed. Virus detection was performed by real-time chain polymerization using the commercial FTlyoTM Respiratory Pathogens 21 kit (Fast Track Diagnostics, Luxembourg) to simultaneously detect the genomes of 18 different viruses and 1 bacterium. Results: Respiratory viruses were detected in all 18 samples, with HRSV identified in all samples, human bocavirus (HBoV) in 7 and human rhinovirus (HRV) in one sample. Coinfections were detected in 38.9% of samples. The HRSV and HBoV co-infection was proven in 33.33% of samples, while the presence of all three viruses in only one sample. Conclusion: The results of this study show the presence of HRSV, HBoV and HRV in bronchoalveolar lavage using multiplex RT-PCR method. Both HRSV and HBoV were the most common viral coinfections. Additional research is needed to more accurately estimate the frequency and distribution of respiratory viruses in children.

Detection of Opportunistic Fungi from the Bronchoalveolar Lavage Specimens of Patients with Pulmonary Diseases.

Opportunistic fungi are a constantly evolving group of pathogens that become active when the immune system is compromised, begin to multiply, and soon overwhelm the weakened immune system. This study was performed to evaluate the number of opportunistic fungi in bronchoalveolar lavage (BAL) samples of patients with pulmonary diseases. After receiving patients' consent and demographic forms, a total of 120 BAL samples were taken by a pulmonary physician. The etiologic agents were identified by standard morphological and molecular methods. Yeast cells were counted on culture media, and direct smears were precisely examined for the presence of yeasts elements, Pneumocystis, and filamentous fungi. In this study, 29 (24.1%) patients showed positive direct smears for yeast elements in their BAL samples. The mean colony count of yeasts was 42,000 (CFU/mL) on culture media. Six (5%) species of filamentous fungi, including three (2.5%) isolates of Penicillium species (P. variabile, P. glabrum, and P. thomii), two (1.67%) Aspergillus species (A. flavus and A. fumigatus), 1 case (0.83%) Pseudallescheria boydii were detected. Seven cases (5.83%) of Pneumocystis cysts were observed in the direct smears stained with Giemsa. Identification of all fungi confirmed by molecular or sequencing methods. Due to the presence of a large number of fungi in the BAL samples and possible physical interference with the selected drugs for treatment, we draw the attention of pulmonologists to this important issue. Rapid diagnosis of fungal infections is essential to optimize treatments and outcomes.

Dynamics of subclinical pneumonia in male dairy calves in relation to antimicrobial therapy and production outcomes.

Quick thoracic ultrasonography (qTUS) is increasingly used as an on-farm method to diagnose clinical and subclinical pneumonia in dairy calves. The primary objective of this prospective cohort study was to describe dynamics of lung consolidation in a purchase-dependent production system for male dairy calves in relation to antimicrobial therapy and respiratory diagnostics. In addition, we studied the association of cured and uncured pneumonia with average daily gain (ADG) and cold carcass weight (CCW). The third objective was to determine the effects of arriving with lung consolidation on the probability of developing chronic unresponsive pneumonia and reduced performance. A total of 295 male dairy calves were intensively followed by qTUS and clinical scoring on 7 strategic occasions (wk 1, 2, 3, 4, 6, 8, and 12) during the production cycle. Of the calves, 17.6% (52/295) arrived with a lung consolidation ≥1 cm. At the first outbreak of respiratory disease (wk 1 after arrival), this incidence had risen to 30.8%. Initial therapy with tulathromycin and subsequently doxycycline appeared ineffective, resulting in a increase to 43.8% of calves having pneumonia in wk 4. At the start of the first outbreak (wk 1), the majority (86.8%) of the pneumonia cases were subclinical. At wk 4, the outbreak became more clinical, and treatment with amoxicillin resulted in a cure risk of 52.7%. Culture and nanopore sequencing diagnostics on nonendoscopic broncho-alveolar lavage (nBAL) samples identified bovine respiratory syncytial virus and Mycoplasma bovis as the dominant agents in the first outbreak. The isolated M. bovis strain showed mutations associated with macrolide resistance. The second outbreak was characterized by a Pasteurella multocida superinfection and isolation of multiple M. bovis strains from nBAL diagnostic testing. Evaluated over the complete observation period, 83.4% of the calves developed consolidations ≥1 cm on qTUS. Of these calves, 53.9% (135/246) were cured by antimicrobial therapy. Chronic pneumonia (≥30 subsequent days of pneumonia) was seen in 13.9% of the animals (n = 41). Calves with uncured or chronic pneumonia had a lower ADG (992 ± 174 g/d and 930 ± 146 g/d, respectively) compared with calves that never developed pneumonia (ADG = 1,103 ± 156 g/d). In contrast, calves that did fully cure trended toward a lower ADG than calves that never developed pneumonia, but differences were no longer significant. Also, the effect of uncured pneumonia was no longer significant for CCW. Calves with lung consolidation upon arrival had a lower ADG (981 ± 159 g/d vs. 1,045 ± 159 g/d) and were more likely to develop chronic pneumonia [odds ratio = 4.2; 95% confidence interval = 2.1-8.6] compared with calves without consolidation upon arrival. Animals with chronic pneumonia, in turn, had a lower CCW than animals without chronic pneumonia (10.3 ± 4.4 kg; 95% confidence interval: 1.6-19.1 kg). This study documents the consequences of subclinical pneumonia upon arrival and pneumonia developed later in the production cycle on production outcomes in a veal calf setting. Both qTUS and nBAL diagnostics provide important information, offering potential for better control and prevention of bovine respiratory disease in dairy calves.

Association Between Bacterial Count and Polymorphonuclear Cells in Uterine Lavage of Postpartum Subclinical Endometritic Cows

This study was conducted to find out the total bacterial count and percentage of polymorphoneutrophils (PMN) cells in uterine lavage samples obtained at a weekly interval from postpartum dairy cows and also to find out the correlation between total bacterial count and PMN cells recovered from the uterus of postpartum dairy cows. Twenty-one postpartum dairy cows were divided into two groups viz. group A (without postpartum complication; n=16) and group B (retained placenta/postpartum metritis; n=5) calving. Cows belonging to the group B were administered antibiotics for 5 days after parturition. Samples for endometrial cytology and total bacterial count were collected aseptically by uterine lavage method at weekly intervals from day 21 to 42 postpartum. Data were statistically analysed by using one-way ANOVA, correlation using Statistical Analysis Software, SAS® 9.2 TS Level version 2M2 for Windows (USA). Based on endometrial cytology, the incidence of sub-clinical endometritis (SCE) recorded was 18.75% in group A and 100% in cows with postpartum complications (Group B). Total bacterial count in the group A animals differed significantly (P<0.05) with the group-B animals. However, the difference between the total bacterial counts in group-A animals and diagnosed negative or positive for SCE did not differ significantly. A highly significant (P<0.05 and P<0.01) correlation was found between bacterial load and PMN cells obtained by uterine lavage technique.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Interest of a Commercialized Pneumocystis jirovecii Quantitative PCR to Discriminate Colonization from Pneumocystis Pneumonia according to the Revised EORTC/MSGERC Criteria.

Quantitative PCR (qPCR) is highly sensitive to diagnose Pneumocystis jirovecii (Pj) pneumonia (PCP). However, differentiating PCP and colonization remains difficult. This study aimed to establish the performances of the commercialized qPCR MycoGENIE® Pj kit (Ademtech) to distinguish PCP and Pj colonization. Patients with a positive Pj qPCR on bronchoalveolar lavage (BAL) or upper respiratory tract (URT) samples were prospectively included between May 2019 and December 2020 at Bordeaux University Hospital. They were classified in “PCP” or “Pj colonization” groups based on the revised EORTC/MSGERC criteria. The two groups’ results were compared; ROC curves were produced to determine the best thresholds. Excluding the low number of HIV-positive subjects, there were 100 PCP (32 BAL, 68 URT) and 70 Pj colonization (34 BAL, 36 URT). Pj loads were significantly higher in PCP compared to Pj colonization group (p ≤ 0.01). The best cut-offs for PCP diagnosis were 31.45 Cq/8275 copies/mL for BAL and 32.33 Cq/8130 copies/mL for URT (sensitivity = 59.4%, 63.3%, specificity = 82.4%, 88.9%, respectively). Fungal load quantification using MycoGENIE® Pj qPCR helps discriminating PCP from colonization, high fungal loads being indicative of probable PCP. Low load results should be interpreted with caution, in accordance with clinical and radiological signs.

A Laboratory-Based Study on Multiple Biomarker Testing in the Diagnosis of COVID-19-Associated Pulmonary Aspergillosis (CAPA): Real-Life Data

(1) Background: Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) raises concerns to contribute to an increased mortality. The incidence of CAPA varies widely within hospitals and countries, partly because of difficulties in obtaining a reliable diagnosis. (2) Methods: Here, we assessed Aspergillus culture-positive and culture-negative respiratory tract specimens via direct fungal microscopy (gold standard) and compared the results with galactomannan enzyme immunoassay (GM-EIA) and Aspergillus PCR. (3) Results: 241 respiratory samples from patients suffering from SARS-CoV-2 pneumonia were evaluated. Results showed both diagnostic tools, Aspergillus PCR and GM-EIA, to be positive or negative displaying a sensitivity of 0.90, a specificity of 0.77, a negative predictive value (NPV) of 0.95, and a positive predictive value (PPV) of 0.58 in Aspergillus sp. culture and microscopic-positive specimens. Non-bronchoalveolar lavage (BAL) samples, obtained within a few days from the same patient, showed a high frequency of intermittent positive or negative GM-EIA or Aspergillus PCR results. Positivity of a single biomarker is insufficient for a proper diagnosis. A broad spectrum of Aspergillus species was detected. (4) Conclusions: Our study highlights the challenges of combined biomarker testing as part of diagnosing CAPA. From the results presented, we highly recommend the additional performance of direct microscopy in respiratory specimens to avoid overestimation of fungal infections by applying biomarkers.

Evaluation of molnupiravir (EIDD-2801) efficacy against SARS-CoV-2 in the rhesus macaque model

Molnupiravir (EIDD-2801) is a prodrug of a ribonucleoside analogue that is currently being used under a US FDA emergency use authorization for the treatment of mild to moderate COVID-19. We evaluated molnupiravir for efficacy as an oral treatment in the rhesus macaque model of SARS-CoV-2 infection. Twenty non-human primates (NHPs) were challenged with SARS-CoV-2 and treated with 75 mg/kg (n = 8) or 250 mg/kg (n = 8) of molnupiravir twice daily by oral gavage for 7 days. The NHPs were observed for 14 days post-challenge and monitored for clinical signs of disease. After challenge, all groups showed a trend toward increased respiration rates. Treatment with molnupiravir significantly reduced viral RNA levels in bronchoalveolar lavage (BAL) samples at Days 7 and 10. Considering the mild to moderate nature of SARS-CoV-2 infection in the rhesus macaque model, this study highlights the importance of monitoring the viral load in the lung as an indicator of pharmaceutical efficacy for COVID-19 treatments. Additionally, this study provides evidence of the efficacy of molnupiravir which supplements the current ongoing clinical trials of this drug.

478. Lung Hybrid Neutrophils and Subsequent Extracellular Traps Are Protective In COVID-19-Associated Pulmonary Aspergillosis

Abstract Background COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus frequently affecting critically ill COVID-19 patients. Pathophysiological insight, key to improve diagnostic and immunomodulatory therapeutic options, is lacking. Methods We performed single-cell RNA sequencing (scRNA-seq) on 37 bronchoalveolar lavage (BAL) samples from 37 critically ill COVID-19 patients. Three groups were defined: patients who did not develop aspergillosis (COVID-19-only, n=22), CAPA patients with sampling < 5 days after CAPA diagnosis (early CAPA, n=6) and CAPA patients with sampling 5-11 days after CAPA diagnosis (late CAPA, n=9). All CAPA patients had probable/proven CAPA according to the 2020 ECMM/ISHAM consensus criteria. Additionally, we assessed neutrophil extracellular trap (NET) levels in a separate cohort of 33 biobanked COVID-19-only BAL samples and 24 early CAPA samples. Results A total of 69008 cells passed quality filtering. CAPA patients had significantly lower BAL neutrophil proportions than COVID-19-only patients, particularly in early CAPA (Fig. 1A). Pseudotime inference revealed two neutrophil trajectories: a regular maturation trajectory, and a trajectory giving rise to “hybrid” neutrophils which express genes encoding proteins with antigen-presenting functions (Fig. 1B). The latter trajectory was dominant in CAPA patients (Fig. 1C). NETosis analyses revealed significantly higher levels of citrullinated histone H3 DNA complexes (H3Cit-DNA) in CAPA patients (Fig. 2A). This explains the low CAPA BAL neutrophil proportions, as neutrophils that underwent NETosis are no longer detected via scRNA-seq. CAPA patients with the lowest H3Cit-DNA levels had significantly decreased survival rates (Fig. 2B). Figure 1:Bronchoalveolar lavage fluid (BALF) neutrophil proportions are significantly decreased in CAPA patients, accompanied by a shift to hybrid neutrophil formation. Panel (A): BALF neutrophil proportions as analyzed by single-cell RNA sequencing using the Seurat R package are significantly lower in CAPA patients compared to COVID-19-only patients. Patients with early CAPA have significantly lower BALF neutrophil proportions than patients with late CAPA. Macrophage/monocyte and epithelial cell proportions are reciprocally increased in CAPA patients compared to COVID-19-only patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (B): Two trajectories are defined using pseudotime inference calculated using the Slingshot R package: a trajectory dominant in COVID-19-only patients with regular maturation of progenitor neutrophils, and a trajectory dominant in CAPA patients with maturation towards a ‘hybrid neutrophil’ state, with neutrophils expression genes encoding proteins with functions in antigen presentation. Subsequently, the hybrid neutrophil proportion is significantly higher in CAPA patients compared to COVID-19-only patients, and is significantly higher in patients with early CAPA than those with late CAPA. The mature neutrophil proportion is reciprocally reduced in CAPA patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Figure 2:Neutrophil extracellular trap (NET) levels are increased in early CAPA and are associated with increased survival in CAPA patients specifically. Panel (A): Myeloperoxidase (MPO) DNA levels were analyzed as measure for general NET-formation, while citrullinated histone H3 bound DNA (H3Cit-DNA) levels were analyzed as more specific PAD4-dependent NET-formation, in BALF samples from early CAPA and COVID-19-only patients. A trend towards higher MPO-DNA levels was found in early CAPA patients, while H3Cit-DNA levels were significantly higher in early CAPA compared to COVID-19-only patients. P-values calculated using Mann-Whitney U test. Panel (B): Kaplan-Meier analysis of patients with NETosis analyses, divided in early CAPA and COVID-19-only patients and subdivided according to H3Cit-DNA levels (cut-off at 20000 ng/mL for early CAPA and at 8000 ng/mL for COVID-19-only). Log-rank test was used to compare survival distributions. For the comparison early CAPA (low H3Cit-DNA) versus early CAPA (high H3Cit-DNA), the log-rank p-value was 0.033. Conclusion CAPA patients display extremely high levels of released NETs in the lower respiratory tract, associated with a shift from the normal neutrophil maturation process towards “hybrid neutrophil” formation, probably upon encountering the fungus. In contrast to high NETosis contributing to mortality in severe COVID-19, CAPA patients likely require these NETs to survive aspergillosis. BAL NET levels hold promise as a tool to guide diagnosis, prognosis and treatment in these patients. Disclosures Simon Feys, MD, Pfizer: Travel support Katrien Lagrou, PharmD, PhD, FUJIFILM Wako: Speaker fee|Gilead: Advisor/Consultant|Gilead: Speaker fee|MRM Health: Advisor/Consultant|MSD: Advisor/Consultant|Pfizer: Speaker fee|Thermo fisher Scientific: Service fee Joost Wauters, MD, PhD, Gilead: Grant/Research Support|Gilead: Speaker's fee, travel support, advisory board|MSD: Grant/Research Support|MSD: Speaker's fee, travel support, posaconazole for interventional trial|Pfizer: Grant/Research Support|Pfizer: Speaker's fee, travel support, advisory board.

544. PICKUP: Pneumonia in the Immunocompromised - Use of the Karius Test for Detection of Undiagnosed Pathogens

Abstract Background Pneumonia is the most common infectious cause of morbidity and excess mortality complicating hematopoietic cell transplantation (HCT) and treatment of hematologic malignancy. Standard bronchoscopic and noninvasive microbiologic testing identify causative pathogens in less than half of cases. The Karius Test, a plasma next-generation sequencing assay of microbial cell-free DNA, may improve diagnostic yield in these patients. Methods Patients with active hematologic malignancy or recent HCT undergoing bronchoscopy for suspected pneumonia were prospectively enrolled in this observational study conducted at 10 United States medical centers. A panel of expert clinicians blinded to Karius Test results reviewed a standardized panel of microbiologic and molecular testing from bronchoalveolar lavage and blood samples for bacterial and fungal testing, nasopharyngeal swab for respiratory viral testing, imaging results, clinical documentation, and any additional microbiologic or molecular testing collected per usual standard of care to adjudicate a probable cause of pneumonia. The panel then adjudicated whether a probable cause of pneumonia or other clinically relevant infection was identified by the Karius Test. Results Between January 3, 2020 and February 4, 2022, 257 patients were enrolled. A planned interim analysis of the first 69 sequentially enrolled patients in the per protocol population was conducted. An adjudicated probable cause of pneumonia was identified by standard care in 18/69 (26%) patients. The Karius Test identified an adjudicated probable cause of pneumonia in 10/51 (20%) patients when no cause of pneumonia was identified by standard care testing. The combination of standard care and the Karius Test together identified a probable cause of pneumonia in 28/69 (41%) patients. At least one additional pathogen adjudicated as a probable cause of pneumonia was identified by the Karius Test in 6/18 (33%) of patients with positive standard care testing. Conclusion The Karius Test notably increased the probability of identifying a pathogenic cause of pneumonia among immunocompromised patients undergoing bronchoscopy. The additive diagnostic value of the Karius Test may significantly enhance management of this common condition. Disclosures Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support Radha Duttagupta, PhD, Karius Inc: Stocks/Bonds Sanjeet S. Dadwal, MD, FACP, FIDSA, AlloVir: Advisor/Consultant|AlloVir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Aseptiscope: Advisor/Consultant|Aseptiscope: Stocks/Bonds|Astellas: Speaker's Bureau|Cidara: Advisor/Consultant|Gilead: Grant/Research Support|Karius: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Speaker's Bureau|Takeda: Speaker's Bureau Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant Ghady Haidar, MD, Karius, Allovir, and AstraZeneca: Grant/Research Support Alfred Luk, MD, Karius: Grant/Research Support Jamie Todd, MD, Altavant Sciences: Advisor/Consultant|AstraZeneca: Grant/Research Support|Boehringer Ingelheim: Grant/Research Support|CareDx: Grant/Research Support|Cellarity: Advisor/Consultant|Natera: Advisor/Consultant Genovefa Papanicolaou, MD, AlloVir: Board Member|AlloVir: Serve as member of DSMC|Amplyx: Board Member|Amplyx: Serve as member of DSMC|Astellas: Advisor/Consultant|Cidara: Advisor/Consultant|CSL Behring: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Investigator for Merck|MSD: Advisor/Consultant|Octapharma: Advisor/Consultant|Octapharma: Board Member|Octapharma: Serve as EAC member|Partners RX: Advisor/Consultant|SymBio: Advisor/Consultant|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Takeda: Investigator for Takeda|Vera: Board Member|Vera: Serve as member of DSMC Elena Nemirovich-Danchenko, MD PhD, Karius: Stocks/Bonds Mona Mughar, BS, Karius: Stocks/Bonds Sudeb Dalai, MD, Karius: Stocks/Bonds Sudeb Dalai, MD, Karius: Stocks/Bonds Yuen Cho, MS, CLS(CA-DPH), Karius: Stocks/Bonds Asim A. Ahmed, MD, Karius: Employee|Karius: Stocks/Bonds Desiree Hollemon, MSN, MPH, Karius: Stocks/Bonds David K. Hong, MD, Janssen Pharmaceutical Companies of Johnson & Johnson: Employee|Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Marla Lay Vaughn, BS, MT(ASCP), Karius: Employee|Karius: Stocks/Bonds Tim Blauwkamp, PhD, Karius: Board Member|Karius: Ownership Interest Zivjena Vucetic, MD, Karius: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds Thomas L. Holland, MD, Aridis: Advisor/Consultant|Lysovant: Advisor/Consultant.

1244: CAN ADMINISTRATION OF HIGH-DOSE HYDROXOCOBALAMIN IMPROVE OUTCOMES IN REFRACTORY SEPTIC SHOCK?

Introduction: Multiple high dose vasopressors are often required to sustain life in critically ill patients but are unfortunately associated with high mortality (>50%) and morbidity. This case discusses the potential benefit in adding high dose IV hydroxocobalamin (B12) therapy (5g) to potentially reduce the use of additional vasopressors and their impact on morbidity and mortality. Description: A 72-year-old immunocompromised man with history of multiple myeloma, refractory to multiple lines of therapy, prior CAR-T treatment with now stable disease was found down, covered in feces. He was admitted to the medical ICU for severe septic shock secondary to pseudomonas bacteremia. The patient progressed to Multiorgan failure requiring mechanical ventilation, broad spectrum antibiotics, and multiple vasopressors at maximum doses. Echocardiogram (echo) showed biventricular heart failure with ejection fraction (EF) reduced from 64% 4 months prior to 25%. The bronchoalveolar lavage sample grew Pseudomonas aeruginosa, and antibiotics were adjusted. He was requiring norepinephrine (NE) at 0.3 mcg/kg/min, vasopressin (vaso) at 0.04 units/min, epinephrine (epi) to 0.3 mcg/kg/min, angiotensin II (ATII) between 10-40 ng/kg/min, and dobutamine (dobut) as high as 12.5 mcg/kg/min for 47 hours before B12 administration. The patient remained hypotensive (MAP < 65 mmHg) with maximal vasopressors. The left hand become cold and discolored, his legs and abdomen became severely mottled. Due to lack of response to therapy a trial of 5g B12 was given. There was improvement in the patient’s blood pressure. NE and ATII were stopped within 24 hours, Epi and vaso stopped within 96 hours, and dobut was weaned gradually. He was extubated on day 12. EF improved to 60% with normal right ventricular function on day 14. The skin gradually returned to normal. The left-hand fingers appeared better perfused with only the tip remaining discolored. Discussion: Bacterial endotoxins released in sepsis induce nitric oxide synthase, activating cGMP, which causes subsequent smooth muscle inhibition and vasodilation. B12 scavenges nitric oxide decreasing vasodilation. Use of B12 as an adjuvant to traditional vasopressor therapy could help reduce morbidity and mortality in septic shock by reducing overall dose/duration of vasopressors.

Adequacy of Bronchoalveolar Lavage in Decreasing Morbidity in Ventilated Neonates

Early-onset pneumonia is part of generalized sepsis that first manifests at or within hours of birth. Late-onset pneumonia usually occurs after 7 days of age, most commonly in neonatal intensive care units among infants who require prolonged endotracheal intubation because of lung disease. Diagnosis is by clinical and laboratory evaluation for sepsis. Treatment is initial broad-spectrum antibiotics changed to organism-specific drugs as soon as possible. Surveillance studies of nosocomial infections in NICU patients indicate that pneumonia comprises 6.8 to 32.3% of nosocomial infections in this setting. Aim: To establish the incidence, aetiology, risk factors, and outcomes associated with pneumonia using BAL sampling technique in neonates. Material and methods: A cross sectional study carried out at neonatal intensive care unit in a tertiary health care centre in Solapur, thus 28 neonates admitted in NICU and requiring mechanical ventilation were studied for period of 2 years. Bronchoalveolar lavage samples were collected using flexible bronchoscope with two sterile mucus extractor. Isolation of &gt;103 colony-forming unit/mL was required for diagnosis. Results: 79% of neonates had culture positive growth and 21% had culture negative growth Most common pathogen isolated was E coli 25%. The organisms isolated were found to have different sensitivity pattern on the culture report and the patients were treated accordingly. Enlisted here are few of the antibiotics which were found to be sensitive. Conclusion: It’s unique and very effective way to isolate the organism causing respiratory pathology and provide antimicrobial therapy according to susceptibility pattern rather than giving broad spectrum antibiotics for a longer duration and time and increasing incidence for drug resistance.

Pharyngeal Inflammation on Positron Emission Tomography/Magnetic Resonance Imaging Before and After Obstructive Sleep Apnea Treatment.

Rationale: There is upper airway inflammation in patients with obstructive sleep apnea (OSA), which reduces with continuous positive airway pressure (CPAP) therapy. Objectives: Validate the use of positron emission tomography (PET)/magnetic resonance imaging (MRI) to quantify metabolic activity within the pharyngeal mucosa of patients with OSA against nasal lavage proteomics and assess the impact of CPAP therapy. Methods: Adults with OSA underwent [18F]-Fluoro-2-deoxy-D-glucose PET/MRI of the neck before and 3 months after initiating CPAP. Nasal lavage samples were collected. Inflammatory protein expression from samples was analyzed using the Olink platform. Upper airway imaging segmentation was performed. Target-to-background ratio (TBRmax) was calculated from target pharyngeal maximum standard uptake values (SUV) and personalized background mean SUV. Most-diseased segment TBRmax was identified per participant at locations with the highest PET avidity. Correlation analysis was performed between baseline TBRmax and nasal lavage proteomics. TBRmax was compared before and after CPAP using linear mixed-effect models. Results: Among 38 participants, the baseline mean age was 46.3 years (standard deviation [SD], 12.5), 21% were female, the mean body mass index was 30.9 kg/m2 (SD, 4.6), and the mean respiratory disturbance index measured by peripheral arterial tonometry was 31 events/h (SD, 16.4). There was a significant positive correlation between pharyngeal mucosa most-diseased segment TBRmax and nasal lavage proteomic inflammation (r = 0.41 [P < 0.001, false discovery rate = 0.002]). Primary analysis revealed a reduction in the most-diseased segment TBRmax after a median of 2.91 months of CPAP therapy (-0.86 [standard error (SE) ± 0.30; P = 0.007]). Stratified analysis by smoking status revealed a significantly decreased most-diseased segment TBRmax after CPAP therapy among never-smokers but not among ever-smokers (-1.01 [SE ± 0.39; P = 0.015] vs. -0.64 [SE ± 0.49; P = 0.201]). Conclusions: CPAP therapy reduces metabolic activity measured by PET/MRI within the upper airway of adults with OSA. Furthermore, PET/MRI measures of upper airway metabolic activity correlate with a noninvasive marker of inflammation (i.e., nasal lavage inflammatory protein expression).

Protein biomarkers in cervicovaginal lavages for detection of endometrial cancer

BackgroundRates of endometrial cancer (EC) are increasing. For a definitive diagnosis, women undergo various time-consuming and painful medical procedures, such as endometrial biopsy with or without hysteroscopy, and dilation and curettage, which may create a barrier to early detection and treatment, particularly for women with inadequate healthcare access. Thus, there is a need to develop robust EC diagnostics based on non- or minimally-invasive sampling. The objective of this study was to quantify a broad range of immuno-oncology proteins in cervicovaginal lavage (CVL) samples and investigate these proteins as predictive diagnostic biomarkers for EC.MethodsOne hundred ninety-two women undergoing hysterectomy for benign or malignant indications were enrolled in this cross-sectional study. Classification of women to four disease groups: benign conditions (n = 108), endometrial hyperplasia (n = 18), low-grade endometrioid carcinoma (n = 53) and other EC subtypes (n = 13) was based on histopathology of biopsy samples collected after the surgery. CVL samples were collected in the operating room during the standard-of-care hysterectomy procedure. Concentrations of 72 proteins in CVL samples were evaluated using multiplex immunoassays. Global protein profiles were assessed using principal component and hierarchical clustering analyses. The relationships between protein levels and disease groups and disease severity were determined using Spearman correlation, univariate and multivariate receiver operating characteristics, and logistic regression analyses.ResultsWomen with EC and benign conditions exhibited distinctive cervicovaginal protein profiles. Several proteins in CVL samples (e.g., an immune checkpoint protein, TIM-3, growth factors, VEGF, TGF-α, and an anti-inflammatory cytokine, IL-10) discriminated EC from benign conditions, particularly, when tested in combinations with CA19–9, CA125, eotaxin, G-CSF, IL-6, MCP-1, MDC, MCP-3 and TRAIL (sensitivity of 86.1% and specificity of 87.9%). Furthermore, specific biomarkers (e.g., TIM-3, VEGF, TGF-α, TRAIL, MCP-3, IL-15, PD-L2, SCF) associated with histopathological tumor characteristics, including histological type and grade, tumor size, presence and depth of myometrial invasion or mismatch repair protein status, implying their potential utility for disease prognosis or monitoring therapies.ConclusionsThis proof-of-principle study demonstrated that cervicovaginal sampling coupled with multiplex immunoassay technology can offer a minimally to non-invasive method for EC detection.

A novel biosensor for ultrasensitive detection of fungal genes

Fungal infections are a rapidly increasing public health problem due to their high morbidity and mortality rates, especially in populations with compromised immune systems. Rapid and accurate diagnosis of these diseases is, therefore, necessary to improve the prognosis of afflicted patients. Unfortunately, current clinical chemistry practice relies on lengthy culturing methods that are insufficient to meet the fast turnaround requirements. Here we present a cost-effective and robust nucleic acid sensor that can identify the presence of histoplasmosis causing fungal genes, in whole blood or bronchoalveolar lavage (BAL) samples, far earlier than current methods. Our novel assay involves the hybridization of target gene sequences with immobilized nucleic acid probes, allowing direct, label-free detection of Hcp100, CBP1, and M antigen genes through electrochemical analysis. The resultant current is attributed to the presence of fungal targets in the sample solution. The assay provides ultra-sensitive detection of DNA molecules with a limit of detection (LOD) values down to 100 aM, sufficient to meet the clinical diagnostic need. In addition, the turnaround time for the sample to result is less than 90 min compared to the current clinical procedure's turnaround time of 3–4 weeks.

GeneXpert analysis of bronchoalveolar lavage (BAL) samples: promising diagnostic modality in patients with smear-negative pulmonary tuberculosis

PurposeThe aim of the study is to assess the diagnostic utility of bronchoalveolar lavage (BAL) samples with GeneXpert test in diagnosis of TB in smear-negative patients.MethodOne-hundred sixty-nine cases were included in this study, as they had a radiological finding suggested to have pulmonary tuberculosis, while the sputum direct smear examination for acid-fast bacilli was negative. All cases prepared to do fiber-optic bronchoscope with bronchoalveolar lavage collection after their consent and post bronchoscope sputum collection for further molecular and microbiological examination.ResultsThe combined diagnostic yield of all bronchoscopy procedures was 15.97% (27 cases of 169) and the final diagnosis of TB cases was established in 16 cases of 169 (9.4%) using BAL culture and in 20 cases of 169 (11.83%) by GeneXpert methods. Post bronchoscope direct sputum examination, culture, and GeneXpert evaluation gave diagnostic yield about 1.1%, 5.5%, and 7.1%, respectively. The specificity of GeneXpert examination of BAL and post bronchoscope sputum was 95.42% and 97.38%, respectively.ConclusionEarly detection of pulmonary tuberculosis using fiber-optic bronchoscope BAL examination and post bronchoscope sputum with GeneXpert method promotes the accurate decision to start antituberculous therapy in patients with smear negative.

First report of a severe nasopulmonary acariasis caused by Orthohalarachne diminuata Doetschman, 1944 (Acari: Halarachnidae) in a captive South American sea lion (Otaria flavescens Shaw, 1800).

Obligatory endoparasitic mites of the genera Halarachne Allman, 1847 and Orthohalarachne Newell, 1947 (Acari: Halarachnidae) parasitize different segments of the respiratory tract of marine mammals, including pinnipeds and sea otters, and infestations can cause asymptomatic to serious respiratory diseases. However, knowledge on biology, pathogenic potential and occurrence of halarachnid mites infesting pinnipeds, especially in captivity, is scarce. A two-year-old South American sea lion (Otaria flavescens Shaw, 1800) male, born and held at the Vienna Zoo, was anesthesized for routine pre-transport examinations, including computed tomography, bronchoalveolar lavage, and blood sampling. During the final phase of general anesthesia, the individual abruptly became apneic and died despite all attempts at resuscitation. At necropsy, 45 highly motile whitish millimeter-sized structures were macroscopically detected in the trachea, bifurcatio tracheae and main bronchi and were identified as adult stages of Orthohalarachne diminuata Doetschman, 1944 following morphological descriptions. After trepanation of the nasal cavity and sinus paranasalis, a total of 407 larval and 3 nymphal specimens distributed in clusters were detected. Macroscopically, sinus mucosa showed hyperemia and multiple petechial hemorrhages. Histopathological analyses of paranasal sinuses revealed mite cross-sections surrounded by sanioserous exudate and epithelial exfoliation. For the first time, O. diminuata was molecularly characterized and phylogenetically analyzed based on its 16S rDNA. Our study constitutes the first record of a severe O. diminuata infestation in captive O. flavescens and one of the few host-parasite records in general. We present clinical data and pathological results, the first scanning electron microscopic images of a O. diminuata larval stage and discuss the etiology of this autochthonous infestation, possible transmission pathways and detrimental effects. Further studies on biology and pathogenic effects of halarachnid mites, as well as on the development of non-invasive sampling techniques are essentially required for a better understanding of (ortho-)halarachnosis in pinnipeds held in zoological gardens.

Immunometabolic profiling of cervicovaginal lavages identifies key signatures associated with adenomyosis.

Adenomyosis is a burdensome gynecologic condition that is associated with pelvic pain, dysmenorrhea, and abnormal uterine bleeding, leading to a negative impact on quality of life; and yet is often left undiagnosed. We recruited 108 women undergoing hysterectomy for benign gynecologic conditions and collected non-invasive cervicovaginal lavage samples for immunometabolic profiling. Patients were grouped according to adenomyosis status. We investigated the levels of 72 soluble immune proteins and >900 metabolites using multiplex immunoassays and an untargeted global metabolomics platform. There were statistically significant alterations in the levels of several immune proteins and a large quantity of metabolites, particularly cytokines related to type II immunity and amino acids, respectively. Enrichment analysis revealed that pyrimidine metabolism, carnitine synthesis, and histidine/histamine metabolism were significantly upregulated pathways in adenomyosis. This study demonstrates utility of non-invasive sampling combined with immunometabolic profiling for adenomyosis detection and a greater pathophysiological understanding of this enigmatic condition.

Prevalence of SARS-CoV-2 Variants in COVID-19 Positive Patients

Objective: Of the existing variants, alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and omicron (B.1.1.529) were defined as variants of concern (VOCs) by World Health Organization. We aimed to retrospectively assess the distribution of VOCs in patients with positive COVID-19 real-time polymerase chain reaction (RT-PCR) test. Methods: Between April 2021 and December 31, 2021, 4260 patients whose clinical samples were sent with a COVID-19 PCR test request were included in the study. Of the patients whose samples were evaluated, 2173 (51%) were male, and 2087 (49%) were female. Viral nucleic acid isolation was performed by treating nasopharyngeal swabs, bronchoalveolar lavage, and tracheal aspirate samples with viral nucleic acid buffer (vNAT). SARS-CoV-2 variants were detected by Bio-Speedy® SARS-CoV-2 Variant Plus kit (Bioeksen AR-GE Technologies, Turkey), which targets variant-specific genome regions only found in B.1.1.7, B.1.351, and P.1 as well as ORF1ab and N gene regions. We used Bio-Speedy® SARS-CoV-2 Emerging Plus kit and CFX96 DX real-time PCR system to detect SARS-Cov-2 variants; N D3L mutation was targeted to detect the alpha variant, SL452R mutation to detect the delta variant, and S E484K mutation, which is widespread, to detect the gamma and the mu variants. Results: SARS-CoV-2 RT-PCR was positive in 773 (18.14%) of 4260 samples, and different SARS-CoV-2 variants were detected in 316 (40.88%) of positive samples. 407 (52.65%) of the patients with real-time polymerase chain reaction (RT-PCR) positive test were women, and the mean age was 41.7 years. While no variant type was detected in 457 (59.12%) of the patients, the alpha variant was detected in 156 (20.18%) and the delta variants in 137 (17.72%) patients. Conclusion: In our study, we found that the alpha variant was the dominant type in April, May, and June, and the delta variant became dominant as of July until the end of the year. Decreased rates of delta variants towards the end of 2021 and an increase in the other variants suggest the omicron variant, widely spread globally. Therefore, we believe that the omicron variant has been our country’s dominant type since November 2021. In conclusion, molecular surveillance studies that are planned in the light of epidemiological data and assessment of the frequency and genomic analyzes of regional and global variants are required.

GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. Preclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte/macrophage infiltration to the lung during influenza A virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of Gpr183 or treatment with a GPR183 antagonist reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single-cell RNA-sequencing data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.