Next-Generation Sequencing for Characterizing Respiratory Tract Virome and Improving Detection of Viral Pathogens in Children With Pneumonia.

BACKGROUND: Pneumonia is typically caused by a variety of pathogenic microorganisms. Traditional research often focuses on the infection of a few microorganisms, whereas metagenomic studies focus on the impact of the bacteriome and mycobiome on respiratory diseases. Reports on the virome characteristics of pediatric pneumonia remain relatively scarce. METHODS: We employed de novo assembly and combined homology- and feature-based methods to characterize the respiratory virome in whole-genome DNA sequencing samples from oropharynx (OP) swabs, nasopharynx (NP) swabs, and bronchoalveolar lavage fluids (BALF) of children with pneumonia. RESULTS: Significant differences were observed in the alpha and beta diversity indexes, as well as in the composition of the oropharyngeal virome, between pneumonia cases and controls. We identified 1137 viral operational taxonomic units (vOTUs) with significant differences, indicating a preference of pneumonia-reduced vOTUs for infecting Prevotella, Neisseria, and Veillonella, whereas pneumonia-enriched vOTUs included polyomavirus, human adenovirus, and phages targeting Staphylococcus, Streptococcus, Granulicatella, and Actinomyces. Comparative analysis revealed higher relative abundances and prevalence rates of pneumonia-enriched OP vOTUs in NP and BALF samples compared to pneumonia-reduced vOTUs. Additionally, virome analysis identified six pediatric patients with severe human adenovirus or polyomavirus infections, five of whom might have been undetected by targeted polymerase chain reaction (PCR)-based testing. CONCLUSIONS: This study offers insights into pediatric pneumonia respiratory viromes, highlighting frequent transmission of potentially pathogenic viruses and demonstrating virome analysis as a valuable adjunct for pathogen detection.

Detection and Genomic Characterization of Torque Teno Virus in Pneumoconiosis Patients in China.

Pneumoconiosis is a common occupational disease that can worsen with accompanying infection. Torque teno virus (TTV) is a prevalent human virus with multiple genotypes that can chronically and persistently infect individuals. However, the prevalence of TTV in pneumoconiosis patients is still unclear. This research aims to detect the presence and prevalence of TTV in the alveolar lavage fluid of pneumoconiosis patients in the Hunan Province of China using PCR. As a result, a 65.5% positive rate (19 out of 29) of TTV was detected. The TTV detection rate varies among different stages of silicosis and different pneumoconiosis patient ages. Nine novel TTV genomes ranging in size from 3719 to 3908 nt, named TTV HNPP1, HNPP2, HNPP3, HNPP4, HNPP5, HNPP6-1, HNPP6-2, HNPP7-1 and HNPP7-2, were identified. A genomic comparison and phylogenetic analysis indicated that these nine TTVs represent five different species with high genetic diversity which belong to the genus Alphatorquevirus. HNPP6-1 and HNPP6-2 belong to TTV3, HNPP5 belongs to TTV13, HNPP1 belongs to TTV24, HNPP4 belongs to TTV20, and the others belong to TTV19. The genomes of TTV HNPP1, HNPP6-1, and HNPP6-2 contain three putative open reading frames (ORFs) coding for proteins, ORF1, ORF2, and ORF3, while the other six TTV genomes contain two ORFs coding for proteins, ORF1 and ORF2. These results provide the first description of TTV epidemiology in pneumoconiosis patients in China. The newly identified TTV genome sequences reveal the high genetic diversity of TTV in pneumoconiosis patients and could contribute to a deeper understanding of TTV retention and infection in humans.

Clinical significance of lower respiratory tract culture within 48 h of admission in patients with viral pneumonia: an observational study.

BACKGROUND: The aim of this retrospective study was to examine the risk factors of positive lower respiratory tract cultures and to investigate whether nosocomial infections are common in patients with positive lower respiratory tract cultures. METHODS: We enrolled 86 patients diagnosed with influenza A-related critical illness who were treated at Fuzhou Pulmonary Hospital of Fujian in China between 1st October 2013 and 31st March 2019. The of admission were used to divide the enrolled patients into two groups. Sputum and bronchoalveolar lavage fluid specimens were collected within 48 h after admission for culture. All samples were cultured immediately after sampling. Nosocomial infections are defined as any symptom or sign of pulmonary infiltration, confirmed by X-ray, after 5 days of admission and positive results from one or more cultures. RESULTS: The average age of this cohort was (54.13 ± 16.52) years. Based on the culture results, Staphylococcus aureus and Candida albicans had the highest positive rates (3.40% (3/86) and 20.90% (18/86), respectively). In patients with positive lower respiratory tract cultures, the incidence of nosocomial infection was 73.30% (22/30) five days after admission. However, the incidence of nosocomial infection was lower (42.80%, 24/56) in patients with negative lower respiratory tract cultures. Hemoptysis, systolic pressure at admission, and blood urea nitrogen level at admission were all independent risk factors for positive lower respiratory tract cultures within 48 h of admission. CONCLUSION: Our data showed that a significant proportion of patients with pneumonia exhibited co-infections with bacteria or fungi within five days of hospital admission. Hemoptysis, systolic pressure, and blood urea nitrogen levels at admission emerged as the key risk factors. These findings underscore the necessity of closely monitoring patients with influenza infection, particularly for positive bacterial or fungal cultures within the initial 48 h of admission.

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunization in the pig lung.

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.

Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

The bacterial and fungal profiles of patients hospitalized with non-COVID-19 lower respiratory tract infections in Wuhan, China, 2019-2021.

AIMS: A severe lockdown occurred in Wuhan during the COVID-19 pandemic, followed by a remission phase in the pandemic's aftermath. This study analyzed the bacterial and fungal profiles of respiratory pathogens in patients hospitalized with non-COVID-19 lower respiratory tract infections (LRTIs) during this period to determine the pathogen profile distributions in different age groups and hospital departments in Wuhan. METHODS AND RESULTS: We collected reports of pathogen testing in the medical records of patients hospitalized with non-COVID-19 LRTI between 2019 and 2021. These cases were tested for bacterial and fungal pathogens using 16S and internal transcribed spacer sequencing methods on bronchoalveolar lavage fluid samples. The study included 1368 cases. The bacteria most commonly identified were Streptococcus pneumoniae (12.50%) and Mycoplasma pneumoniae (8.33%). The most commonly identified fungi were Aspergillus fumigatus (2.49%) and Pneumocystis jirovecii (1.75%). Compared to 2019, the S. pneumoniae detection rates increased significantly in 2021, and those of M. pneumoniae decreased. Streptococcus pneumoniae was detected mainly in children. The detection rates of almost all fungi were greater in the respiratory Intensive Care Unit compared to respiratory medicine. Streptococcus pneumoniae and M. pneumoniae were detected more frequently in the pediatric department. CONCLUSIONS: Before and after the COVID-19 outbreak, a change in the common pathogen spectrum was detected in patients with non-COVID-19 in Wuhan, with the greatest change occurring among children. The major pathogens varied by the patient's age and the hospital department.

Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa.

BACKGROUND: Most of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host. METHODS: Here, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase). RESULTS: Interestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1ß. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host. CONCLUSIONS: The phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

Comparative single-cell analysis reveals IFN-γ as a driver of respiratory sequelae after acute COVID-19.

Postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) represent an urgent public health challenge and are estimated to affect more than 60 million individuals globally. Although a growing body of evidence suggests that dysregulated immune reactions may be linked with PASC symptoms, most investigations have primarily centered around blood-based studies, with few focusing on samples derived from affected tissues. Furthermore, clinical studies alone often provide correlative insights rather than causal mechanisms. Thus, it is essential to compare clinical samples with relevant animal models and conduct functional experiments to understand the etiology of PASC. In this study, we comprehensively compared bronchoalveolar lavage fluid single-cell RNA sequencing data derived from clinical PASC samples and a mouse model of PASC. This revealed a pro-fibrotic monocyte-derived macrophage response in respiratory PASC, as well as abnormal interactions between pulmonary macrophages and respiratory resident T cells, in both humans and mice. Interferon-γ (IFN-γ) emerged as a key node mediating the immune anomalies in respiratory PASC. Neutralizing IFN-γ after the resolution of acute SARS-CoV-2 infection reduced lung inflammation and tissue fibrosis in mice. Together, our study underscores the importance of performing comparative analysis to understand the cause of PASC and suggests that the IFN-γ signaling axis might represent a therapeutic target.

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.

A single-center, retrospective study of hospitalized patients with lower respiratory tract infections: clinical assessment of metagenomic next-generation sequencing and identification of risk factors in patients.

INTRODUCTION: Lower respiratory tract infections(LRTIs) in adults are complicated by diverse pathogens that challenge traditional detection methods, which are often slow and insensitive. Metagenomic next-generation sequencing (mNGS) offers a comprehensive, high-throughput, and unbiased approach to pathogen identification. This retrospective study evaluates the diagnostic efficacy of mNGS compared to conventional microbiological testing (CMT) in LRTIs, aiming to enhance detection accuracy and enable early clinical prediction. METHODS: In our retrospective single-center analysis, 451 patients with suspected LRTIs underwent mNGS testing from July 2020 to July 2023. We assessed the pathogen spectrum and compared the diagnostic efficacy of mNGS to CMT, with clinical comprehensive diagnosis serving as the reference standard. The study analyzed mNGS performance in lung tissue biopsies and bronchoalveolar lavage fluid (BALF) from cases suspected of lung infection. Patients were stratified into two groups based on clinical outcomes (improvement or mortality), and we compared clinical data and conventional laboratory indices between groups. A predictive model and nomogram for the prognosis of LRTIs were constructed using univariate followed by multivariate logistic regression, with model predictive accuracy evaluated by the area under the ROC curve (AUC). RESULTS: (1) Comparative Analysis of mNGS versus CMT: In a comprehensive analysis of 510 specimens, where 59 cases were concurrently collected from lung tissue biopsies and BALF, the study highlights the diagnostic superiority of mNGS over CMT. Specifically, mNGS demonstrated significantly higher sensitivity and specificity in BALF samples (82.86% vs. 44.42% and 52.00% vs. 21.05%, respectively, p < 0.001) alongside greater positive and negative predictive values (96.71% vs. 79.55% and 15.12% vs. 5.19%, respectively, p < 0.01). Additionally, when comparing simultaneous testing of lung tissue biopsies and BALF, mNGS showed enhanced sensitivity in BALF (84.21% vs. 57.41%), whereas lung tissues offered higher specificity (80.00% vs. 50.00%). (2) Analysis of Infectious Species in Patients from This Study: The study also notes a concerning incidence of lung abscesses and identifies Epstein-Barr virus (EBV), Fusobacterium nucleatum, Mycoplasma pneumoniae, Chlamydia psittaci, and Haemophilus influenzae as the most common pathogens, with Klebsiella pneumoniae emerging as the predominant bacterial culprit. Among herpes viruses, EBV and herpes virus 7 (HHV-7) were most frequently detected, with HHV-7 more prevalent in immunocompromised individuals. (3) Risk Factors for Adverse Prognosis and a Mortality Risk Prediction Model in Patients with LRTIs: We identified key risk factors for poor prognosis in lower respiratory tract infection patients, with significant findings including delayed time to mNGS testing, low lymphocyte percentage, presence of chronic lung disease, multiple comorbidities, false-negative CMT results, and positive herpesvirus affecting patient outcomes. We also developed a nomogram model with good consistency and high accuracy (AUC of 0.825) for predicting mortality risk in these patients, offering a valuable clinical tool for assessing prognosis. CONCLUSION: The study underscores mNGS as a superior tool for lower respiratory tract infection diagnosis, exhibiting higher sensitivity and specificity than traditional methods.

The diagnostic value of metagenomics next-generation sequencing in HIV-infected patients with suspected pulmonary infections.

Background: Traditional microbiological detection methods used to detect pulmonary infections in people living with HIV (PLHIV) are usually time-consuming and have low sensitivity, leading to delayed treatment. We aimed to evaluate the diagnostic value of metagenomics next-generation sequencing (mNGS) for microbial diagnosis of suspected pulmonary infections in PLHIV. Methods: We retrospectively analyzed PLHIV who were hospitalized due to suspected pulmonary infections at the sixth people hospital of Zhengzhou from November 1, 2021 to June 30, 2022. Bronchoalveolar lavage fluid (BALF) samples of PLHIV were collected and subjected to routine microbiological examination and mNGS detection. The diagnostic performance of the two methods was compared to evaluate the diagnostic value of mNGS for unknown pathogens. Results: This study included a total of 36 PLHIV with suspected pulmonary infections, of which 31 were male. The reporting period of mNGS is significantly shorter than that of CMTs. The mNGS positive rate of BALF samples in PLHIV was 83.33%, which was significantly higher than that of smear and culture (44.4%, P<0.001). In addition, 11 patients showed consistent results between the two methods. Futhermore, mNGS showed excellent performance in identifying multi-infections in PLHIV, and 27 pathogens were detected in the BALF of 30 PLHIV by mNGS, among which 15 PLHIV were found to have multiple microbial infections (at least 3 pathogens). Pneumocystis jirovecii, human herpesvirus type 5, and human herpesvirus type 4 were the most common pathogen types. Conclusions: For PLHIV with suspected pulmonary infections, mNGS is capable of rapidly and accurately identifying the pathogen causing the pulmonary infection, which contributes to implement timely and accurate anti-infective treatment.

Evaluation of Alinity m CMV assay performance for detecting CMV in plasma, cerebrospinal fluid, and bronchoalveolar lavage specimens.

Accurate detection and quantification of cytomegalovirus (CMV) is crucial to preventing adverse outcomes in immunocompromised individuals. Current assays were developed for use with plasma specimens, but CMV may be present in bronchoalveolar lavage (BAL) fluid and cerebrospinal fluid (CSF). We evaluated the performance of the Abbott Alinity m CMV assay compared to the Abbott RealTime CMV assay for quantification of CMV in plasma, BAL, and CSF specimens. To evaluate clinical performance, 190 plasma, 78 BAL, and 20 CSF specimens were tested with the Alinity m assay and compared to the RealTime assay. The Alinity m CMV assay showed high precision (SD <0.01 to 0.13) for all 3 specimen types. Clincal plasma and BAL specimens with quantifiable CMV DNA demonstrated strong correlation to RealTime CMV assay results (r2 = 0.9779 for plasma, r2 = 0.9373 for BAL). The Alinity m CMV assay may be useful for quantification of CMV in plasma, BAL, and CSF specimens.

Clinical findings and outcome predictors for multinodular pulmonary fibrosis in horses: 46 cases (2009-2019).

BACKGROUND: Prognostic indicators for equine multinodular pulmonary fibrosis (EMPF), an interstitial fibrosing lung disease, are poorly described. HYPOTHESIS/OBJECTIVES: Describe diagnostic findings and outcome predictors for EMPF. ANIMALS: Forty-six adult horses with EMPF. METHODS: Retrospective multicenter case series from 2009 to 2019. Radiographic (n = 27) and ultrasonographic studies (n = 19) from EMPF horses and bronchoalveolar lavage fluid (BALF) cytology from 6 EMPF and 13 asthma cases were independently reviewed and blinded to diagnosis and outcome. Associations between predictor variables and survival were assessed by predictor screening followed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Primary clinical findings were weight loss (36/46, 78%), increased respiratory effort (33/46, 72%), tachypnea (32/46, 70%), and fever (18/46, 39%). Macrophage atypia was seen in more EMPF than asthmatic horse BALF (67% vs. 8%; P = .02). Equine herpesvirus 5 (EHV-5) was detected in 24 of 30 (80%) and hyperfibrinogenemia in 25 of 28 (89%) cases. Twenty-seven of 46 horses (59%) and 11 of 45 (24%) survived to discharge and to 3 months, respectively. Three-month survival was associated with lower median (range) respiratory rates (30 [24-36] vs. 41 [30-60] breaths per minute; P = .04), and higher BALF lymphocyte:neutrophil ratios (4.7 [1.4-22] vs. 0.47 [0.11-1.9]; P = .01) and blood lymphocyte counts (1.25 [0.93-2.55] vs. 0.90 [0.70-1.24] × 109/L; P = .03). Imaging findings, EHV-5 detection, and corticosteroid treatment were not associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Fever is not a sensitive clinical sign of EMPF. Diagnostic testing should be pursued for horses with increased respiratory rate and effort and weight loss. The prognosis for EMPF horses is poor. Corticosteroid treatment does not improve 3-month survival.

Kaposi sarcoma herpesvirus viral load in bronchoalveolar lavage as a diagnostic marker for pulmonary Kaposi sarcoma.

OBJECTIVE: Kaposi sarcoma is a vascular tumor that affects the pulmonary system. However, the diagnosis of airway lesions suggestive of pulmonary Kaposi sarcoma (pKS) is reliant on bronchoscopic visualization. We evaluated the role of Kaposi sarcoma herpesvirus (KSHV) viral load in bronchoalveolar lavage (BAL) as a diagnostic biomarker in patients with bronchoscopic evidence of pKS and evaluated inflammatory cytokine profiles in BAL and blood samples. DESIGN: In this retrospective study, we evaluated KSHV viral load and cytokine profiles within BAL and blood samples in patients who underwent bronchoscopy for suspected pKS between 2016 and 2021. METHODS: KSHV viral load and cytokine profiles were obtained from both the circulation and BAL samples collected at the time of bronchoscopy to evaluate compartment-specific characteristics. BAL was centrifuged and stored as cell pellets and KSHV viral load was measured using primers for the KSHV K6 gene regions. RESULTS: We evaluated 38 BAL samples from 32 patients (30 with HIV co-infection) of whom 23 had pKS. In patients with airway lesions suggestive of pKS, there was higher KSHV viral load (median 3188 vs. 0 copies/10 6 cell equivalent; P  = 0.0047). A BAL KSHV viral load cutoff of 526 copies/10 6 cells had a sensitivity of 72% and specificity of 89% in determining lesions consistent with pKS. Those with pKS also had higher IL-1ß and IL-8 levels in BAL. The 3-year survival rate for pKS patients was 55%. CONCLUSION: KSHV viral load in BAL shows potential for aiding in pKS diagnosis. Patients with pKS also have evidence of cytokine dysregulation in BAL.

Quantitative SARS-CoV-2 RT-PCR and Bronchoalveolar Cytokine Concentrations Redefine the COVID-19 Phenotypes in Critically Ill Patients.

RATIONALE: Recent studies suggest that both hypo- and hyperinflammatory acute respiratory distress syndrome (ARDS) phenotypes characterize severe COVID-19-related pneumonia. The role of lung Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV-2) viral load in contributing to these phenotypes remains unknown. OBJECTIVES: To redefine COVID-19 ARDS phenotypes when considering quantitative SARS-CoV-2 RT-PCR in the bronchoalveolar lavage of intubated patients. To compare the relevance of deep respiratory samples versus plasma in linking the immune response and the quantitative viral loads. METHODS: Eligible subjects were adults diagnosed with COVID-19 ARDS who required mechanical ventilation and underwent bronchoscopy. We recorded the immune response in the bronchoalveolar lavage and plasma and the quantitative SARS-CoV-2 RT-PCR in the bronchoalveolar lavage. Hierarchical clustering on principal components was applied separately on the 2 compartments' datasets. Baseline characteristics were compared between clusters. MEASUREMENTS AND RESULTS: Twenty subjects were enrolled between August 2020 and March 2021. Subjects underwent bronchoscopy on average 3.6 days after intubation. All subjects were treated with dexamethasone prior to bronchoscopy, 11 of 20 (55.6%) received remdesivir and 1 of 20 (5%) received tocilizumab. Adding viral load information to the classic 2-cluster model of ARDS revealed a new cluster characterized by hypoinflammatory responses and high viral load in 23.1% of the cohort. Hyperinflammatory ARDS was noted in 15.4% of subjects. Bronchoalveolar lavage clusters were more stable compared to plasma. CONCLUSIONS: We identified a unique group of critically ill subjects with COVID-19 ARDS who exhibit hypoinflammatory responses but high viral loads in the lower airways. These clusters may warrant different treatment approaches to improve clinical outcomes.

Effect of acyclovir therapy on the outcome of mechanically ventilated patients with lower respiratory tract infection and detection of herpes simplex virus in bronchoalveolar lavage: protocol for a multicentre, randomised controlled trial (HerpMV).

INTRODUCTION: Herpes simplex virus (HSV) is frequently detected in the respiratory tract of mechanically ventilated patients and is associated with a worse outcome. The aim of this study is to determine whether antiviral therapy in HSV-positive patients improves outcome. METHODS AND ANALYSIS: Prospective, multicentre, open-label, randomised, controlled trial in parallel-group design. Adult, mechanically ventilated patients with pneumonia and HSV type 1 detected in bronchoalveolar lavage (≥105 copies/mL) are eligible for participation and will be randomly allocated (1:1) to receive acyclovir (10 mg/kg body weight every 8 hours) for 10 days (or until discharge from the intensive care unit if earlier) or no intervention (control group). The primary outcome is mortality measured at day 30 after randomisation (primary endpoint) and will be analysed with Cox mixed-effects model. Secondary endpoints include ventilator-free and vasopressor-free days up to day 30. A total of 710 patients will be included in the trial. ETHICS AND DISSEMINATION: The trial was approved by the responsible ethics committee and by Germany's Federal Institute for Drugs and Medical Devices. The clinical trial application was submitted under the new Clinical Trials Regulation through CTIS (The Clinical Trials Information System). In this process, only one ethics committee, whose name is unknown to the applicant, and Germany's Federal Institute for Drugs and Medical Devices are involved throughout the entire approval process. Results will be published in a journal indexed in MEDLINE and CTIS. With publication, de-identified, individual participant data will be made available to researchers. TRIAL REGISTRATION NUMBER: NCT06134492.

An in silico analysis identifies drugs potentially modulating the cytokine storm triggered by SARS-CoV-2 infection.

The ongoing COVID-19 pandemic is one of the biggest health challenges of recent decades. Among the causes of mortality triggered by SARS-CoV-2 infection, the development of an inflammatory "cytokine storm" (CS) plays a determinant role. Here, we used transcriptomic data from the bronchoalveolar lavage fluid (BALF) of COVID-19 patients undergoing a CS to obtain gene-signatures associated to this pathology. Using these signatures, we interrogated the Connectivity Map (CMap) dataset that contains the effects of over 5000 small molecules on the transcriptome of human cell lines, and looked for molecules which effects on transcription mimic or oppose those of the CS. As expected, molecules that potentiate immune responses such as PKC activators are predicted to worsen the CS. In addition, we identified the negative regulation of female hormones among pathways potentially aggravating the CS, which helps to understand the gender-related differences in COVID-19 mortality. Regarding drugs potentially counteracting the CS, we identified glucocorticoids as a top hit, which validates our approach as this is the primary treatment for this pathology. Interestingly, our analysis also reveals a potential effect of MEK inhibitors in reverting the COVID-19 CS, which is supported by in vitro data that confirms the anti-inflammatory properties of these compounds.

Long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 are differentially expressed in severe COVID-19 patients: An integrated single-cell analysis.

Hyperactive and damaging inflammation is a hallmark of severe rather than mild Coronavirus disease 2019 (COVID-19). To uncover key inflammatory differentiators between severe and mild COVID-19, we applied an unbiased single-cell transcriptomic analysis. We integrated two single-cell RNA-seq datasets with COVID-19 patient samples, one that sequenced bronchoalveolar lavage (BAL) cells and one that sequenced peripheral blood mononuclear cells (PBMCs). The combined cell population was then analyzed with a focus on genes associated with disease severity. The immunomodulatory long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 were highly differentially expressed between mild and severe patients in multiple cell types. Within those same cell types, the concurrent detection of other severity-associated genes involved in cellular stress response and apoptosis regulation suggests that the pro-inflammatory functions of these lncRNAs may foster cell stress and damage. Thus, NEAT1 and MALAT1 are potential components of immune dysregulation in COVID-19 that may provide targets for severity related diagnostic measures or therapy.

Inhibitory effects and mechanisms of the anti-covid-19 traditional Chinese prescription, Keguan-1, on acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Keguan-1, a new traditional Chinese medicine (TCM) prescription contained seven Chinese herbs, is developed to treat coronavirus disease 19 (COVID-19). The first internationally registered COVID-19 randomised clinical trial on integrated therapy demonstrated that Keguan-1 significantly reduced the incidence of ARDS and inhibited the severe progression of COVID-19. AIM OF THE STUDY: To investigate the protective mechanism of Keguan-1 on ARDS, a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model was used to simulate the pathological state of ARDS in patients with COVID-19, focusing on its effect and mechanism on ALI. MATERIALS AND METHODS: Mice were challenged with LPS (2 mg/kg) by intratracheal instillation (i.t.) and were orally administered Keguan-1 (low dose, 1.25 g/kg; medium dose, 2.5 g/kg; high dose, 5 g/kg) after 2 h. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h and 24 h after i.t. administration of LPS. The levels of inflammatory factors tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (KC or mCXCL1), macrophage inflammatory protein 2 (MIP2 or mCXCL2), angiotensin II (Ang II), and endothelial cell junction-associated proteins were analysed using ELISA or western blotting. RESULTS: Keguan-1 improved the survival rate, respiratory condition, and pathological lung injury; decreased the production of proinflammatory factors (TNF-α, IL-6, IL-1ß, KC, and MIP2) in BALF and the number of neutrophils in the lung tissues; and ameliorated inflammatory injury in the lung tissues of the mice with LPS-induced ALI. Keguan-1 also reduced the expression of Ang II and the adhesion molecule ICAM-1; increased tight junction proteins (JAM-1 and claudin-5) and VE-cadherin expression; and alleviated pulmonary vascular endothelial injury in LPS-induced ALI. CONCLUSION: These results demonstrate that Keguan-1 can improve LPS-induced ALI by reducing inflammation and pulmonary vascular endothelial injury, providing scientific support for the clinical treatment of patients with COVID-19. Moreover, it also provides a theoretical basis and technical support for the scientific use of TCMs in emerging infectious diseases.

Respiratory viruses associated with severe mechanically ventilated pneumonia in children.

The burden of pneumonia, especially that caused by respiratory viruses, is markedly high in the pediatric age group. This study aimed to assess viral agents causing severe pneumonia among mechanically ventilated patients. Nonbronchoscopic bronchoalveolar lavage was performed for pediatric patients having severe pneumonia indicated for mechanical ventilation to be tested with a multiplex PCR immediate diagnosis of their etiologic pathogen. Among the 75 patients recruited, viral agents were detected in 73.4% of cases. Rhinovirus and respiratory syncytial virus (RSV) were the most common viruses detected in 32.1% and 29.5%, respectively. The rate of viral infection showed a clear increased incidence in the winter season. The mortality rate among viral-associated severe pneumonia reached 56.36%. Odds of mortality increased threefolds in presence of comorbid conditions and 10-folds with congenital heart disease. The study demonstrated the neglected importance of rhinovirus besides RSV in causing severe critical pneumonia in the pediatric age.