Background To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in immunocompromised patients for the diagnosis of suspected pneumonia in comparison with that of conventional microbiological tests (CMTs). Methods Sixty-nine immunocompromised patients with suspected pneumonia received both CMTs and mNGS of BALF were analyzed retrospectively. The diagnostic value was compared between CMTs and mNGS, using the clinical composite diagnosis as the reference standard. Results Sixty patients were diagnosed of pneumonia including fifty-two patients with identified pathogens and eight patients with probable pathogens. Taking the composite reference standard as a gold standard, 42 pathogens were identified by CMTs including nine bacteria, 17 fungi, 8 virus, 6 Mycobacterium Tuberculosis, and two Legionella and 19(45%) of which were detected by BALF culture. As for mNGS, it identified 76 pathogens including 20 bacteria, 31 fungi, 14 virus, 5 Mycobacterium Tuberculosis, four Legionella and two Chlamydia psittaci. The overall detection rate of mNGS for pathogens were higher than that of CMTs. However, a comparable diagnostic accuracy of mNGS and CMTs were found for bacterial and viral infections. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs (78% vs. 57%, P < 0.05), which mainly because of the high sensitivity of mNGS in patients with Pneumocystis jirovecii pneumonia (PJP) (100% vs. 28%, P < 0.05). Nineteen patients were identified as pulmonary co-infection, mNGS test showed a higher detection rate and broader spectrum for pathogen detection than that of CMTs in co-infection. Moreover, Pneumocystis jirovecii was the most common pathogen in co-infection and mNGS have identified much more co-pathogens of PJP than CMTs. Conclusions mNGS of BALF improved the microbial detection rate of pathogens and exhibited remarkable advantages in detecting PJP and identifying co-infection in immunocompromised patients.

BackgroundAcute respiratory distress syndrome (ARDS) is a challenging clinical problem. Discovering the potential metabolic alterations underlying the ARDS is important to identify novel therapeutic target and improve the prognosis. Serum and urine metabolites can reflect systemic and local changes and could help understanding metabolic characterization of community-acquired pneumonia (CAP) with ARDS.MethodsClinical data of patients with suspected CAP at the First Affiliated Hospital of Wenzhou Medical University were collected from May 2020 to February 2021. Consecutive patients with CAP were enrolled and divided into two groups: CAP with and without ARDS groups.1H nuclear magnetic resonance-based metabolomics analyses of serum and urine samples were performed before and after treatment in CAP with ARDS (n = 43) and CAP without ARDS (n = 45) groups. Differences metabolites were identifed in CAP with ARDS. Furthermore, the receiver operating characteristic (ROC) curve was utilized to identify panels of significant metabolites for evaluating therapeutic effects on CAP with ARDS. The correlation heatmap was analyzed to further display the relationship between metabolites and clinical characteristics.ResultsA total of 20 and 42 metabolites were identified in the serum and urine samples, respectively. Serum metabolic changes were mainly involved in energy, lipid, and amino acid metabolisms, while urine metabolic changes were mainly involved in energy metabolism. Elevated levels of serum 3-hydroxybutyrate, lactate, acetone, acetoacetate, and decreased levels of serum leucine, choline, and urine creatine and creatinine were detected in CAP with ARDS relative to CAP without ARDS. Serum metabolites 3-hydroxybutyrate, acetone, acetoacetate, citrate, choline and urine metabolite 1-methylnicotinamide were identified as a potential biomarkers for assessing therapeutic effects on CAP with ARDS, and with AUCs of 0.866 and 0.795, respectively. Moreover, the ROC curve analysis revealed that combined characteristic serum and urine metabolites exhibited a better classification system for assessing therapeutic effects on CAP with ARDS, with a AUC value of 0.952. In addition, differential metabolites strongly correlated with clinical parameters in patients with CAP with ARDS.ConclusionsSerum- and urine-based metabolomics analyses identified characteristic metabolic alterations in CAP with ARDS and might provide promising circulatory markers for evaluating therapeutic effects on CAP with ARDS.

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Background: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in immunocompromised patients for the diagnosis of suspected pneumonia in comparation to that of conventional microbiological tests (CMTs).Methods: Sixty-nine immunocompromised patients with suspected pneumonia received both CMTs and mNGS of BALF were analyzed retrospectively. The diagnostic value was compared between CMTs and mNGS, using the clinical composite diagnosis as the reference standard. Results: Sixty patients were diagnosed of pneumonia including fifty-two patients with identified pathogens and eight patients with probable pathogens. The overall detection rate of mNGS for pathogens were higher than that of CMTs. However, a comparable diagnostic accuracy of mNGS and CMTs were found for bacterial and viral infections. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs (78% vs. 54%, P＜0.05), which mainly because of the high sensitivity of mNGS in patients with Pneumocystis jirovecii pneumonia (PJP) (100% vs. 28%, P＜0.05). Among fifty-two patients with definite pathogens, nineteen patients (37%) were identified as pulmonary mixed infection, mNGS test showed a higher detection rate and broader spectrum for pathogen detection than that of CMTs in mixed infection. Moreover, Pneumocystis jirovecii was the most common pathogen in mix infection and mNGS have identified much more co-pathogens of PJP than CMTs.Conclusions: mNGS of BALF improved the microbial detection rate of pathogens and exhibited remarkable advantages in detecting PJP and identifying mixed infections in immunocompromised patients.

Background: The currently available treatment methods are ineffective in reducing mortality or improving outcomes in acute lung injury (ALI). The activation of protein kinase C alpha (PKCα) has recently been implicated in ALI development. We explored the potential therapeutic outcomes of PKCα inhibition in cases of ALI and to elucidate the related mechanisms.Methods: Indexes of lung inflammation and injury were examined in lipopolysaccharide (LPS)-treated C57BL/6J mice (male) and macrophages after pretreatment with a PKCα inhibitor. Tissues were collected to assess lung injury by hematoxylin and eosin (H&E) staining. Bronchoalveolar lavage fluid was used to measure the pulmonary edema, hyperinflammatory response, and oxidative stress by bicinchoninic acid (BCA) method and enzyme-linked immunosorbent assay (ELISA). We tested the effect of PKCα inhibition on LPS-induced proliferation, cytotoxicity, oxidative damage, and the release of inflammatory cytokines in macrophages using the Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) cytotoxicity assay kit, flow cytometry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and ELISA.The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway related proteins were detected by Western blot, immunohistochemistry (IHC), and immunofluorescence staining. Results:We observed that LPS upregulated PKCα phosphorylation, induced a hyperinflammatory response, and caused lung injury. However, PKCα inhibition effectively attenuated the changes caused by LPS. Moreover, we confirmed that inhibiting PKCα weakened the activity of the NF-κB pathway under LPS-induced ALI. These findings indicated that inhibition of PKCα is protective against LPS-induced hyperinflammatory response in ALI, this effect is likely to attributed to the downregulation of NF-κB signaling pathways. Conclusions:The results showed that PKCα inhibition could attenuate ALI which may closely related to its anti-inflammatory and anti-oxidative effects.