OBJECTIVES:The global spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) involves evolutionary convergence. This study is to characterize the chromosomal integration of virulence plasmid fragments in a newly emerged CR-hvKP sequence type 2237 (ST2237) clone and to reconstruct its evolutionary history. METHODS:Seven clinical ST2237 isolates were collected from patients at Beijing Bo'ai Hospital, China, between November 2019 and June 2020. Isolates underwent short-read and nanopore long-read sequencing; complete genomes were assembled and annotated. All publicly available ST2237 genomes (n = 67) were incorporated for comparative, phylogenetic and Bayesian evolutionary analyses. RESULTS:The seven isolates were recovered from six patients in a single ward of Bo'ai Hospital, exhibited 1-17 pairwise single nucleotide polymorphisms and displayed a multidrug-resistant phenotype, indicating ongoing clonal dissemination. A ∼188 kb virulence plasmid fragment carrying iucABCD-iutA, rmpA2 and multiple resistance genes, flanked by IS26 elements, was chromosomally integrated in six of the seven isolates. Across Chinese ST2237 genomes, 95% harboured this integration, with minor rearrangements and occasional virulence gene deletions. The most recent common ancestor was dated to approximately 2012, with five major clades subsequently evolving and inter-provincial spreading across northern, central and western China. CONCLUSIONS:IS26-mediated chromosomal integration of large virulence-resistance modules represents a stable evolutionary strategy in this ST2237 CR-hvKP clone. The nosocomial cluster observed in the hospital and its dissemination to multiple provinces highlight the need for continuous nationwide genomic surveillance of this high-risk clone.

Background: Rapid molecular testing enhances pathogen detection in community-acquired pneumonia (CAP), yet its impact on clinical outcomes remains uncertain. We evaluated whether early precision treatment (PT) guided by evolving molecular diagnostics improves in-hospital mortality, and sought to identify specific patient subgroups that derive the greatest survival benefit. Methods: The multicentre retrospective CCAPP cohort study (June 2005–July 2024) included 2430 adults hospitalised with CAP across four general hospitals in Fujian Province, China, yielding 1904 patients with complete data for final analysis. Patients were categorized by diagnostic strategy: limited-target (LND; n=794; 11 bacterial and atypical pathogens) or diverse-target (DND; n=678; 33 bacterial, atypical, fungal, and viral pathogens) nucleic acid detection, or metagenomic next-generation sequencing (mNGS; n=432). On the basis of whether empirical anti-infective regimens administered within 3 days of admission covered clinically relevant pathogens, patients were classified as PT, non-precision treatment (NP), or no pathogen detected (NPD), and were further stratified into respiratory failure (RF) and non-respiratory failure (nRF) subgroups. Primary outcome was in-hospital mortality; secondary outcomes included length of stay and hospital costs. Confounding was addressed via 1:1:1 triad matching (PT:NP:NPD) in the DND (n=318) and mNGS (n=111) cohorts, analysed using conditional logistic regression and Kaplan-Meier methods. Findings: Pathogen positivity was 23·8% (LND), 60·0% (DND), and 32·6% (mNGS). PT was associated with significantly lower mortality compared with NP exclusively in the mNGS cohort, observed in both the overall population (3·4% vs 18·1%, p<0·001) and the RF subgroup (6·5% vs 34·1%, p=0·002). After matching, logistic regression in the DND cohort revealed that PT mortality benefit was strictly confined to the RF subgroup (OR 0·17, 95% CI 0·01–0·98; p=0·049; overall OR 0·43, p=0·220). Distinctly, PT in the mNGS cohort was associated with markedly lower mortality across both the overall population (OR 0·20, 95% CI 0·05–0·88; p=0·034) and the RF subgroup. Survival analyses corroborated this divergence: survival benefits for PT were consistent across the entire mNGS cohort (overall p=0·027; RF p=0·022), whereas DND-era benefits remained restricted to RF patients (p=0·040). PT did not significantly reduce length of stay or costs compared with non-PT. Interpretation: For hospitalised CAP, standard empirical therapy suffices for nRF patients, as PT yields no additional survival benefit, nor does it decrease length of stay or total costs. Conversely, RF patients derive substantial mortality reduction from PT guided by broad-spectrum diagnostics. While mNGS maximizes this protective effect, DND serves as a pragmatic alternative in resource-constrained settings.

Staphylococcus lugdunensis, a coagulase-negative staphylococcus, has emerged as an opportunistic pathogen causing severe infections. It exhibits genetic diversity, with multiple sequence types, antimicrobial resistance (AMR) genes, and virulence factors. This study aimed to explore its genomic features and clinical implications. Six clinical isolates from Fujian, Beijing, and Wuhan were sequenced using Illumina and Oxford Nanopore platforms, and antimicrobial susceptibility was tested. Other 139 genomes were retrieved from GenBank. Multi-locus sequence typing, phylogenetic and recombination analyses, resistance/virulence gene detection, plasmid replicon identification, and staphylococcal cassette chromosome mec (SCCmec) typing were performed. Among 144 genomes, 19 sequence types (STs) were grouped into five clonal complexes (CCs), with CC3 (notably ST3) and ST27 predominating. Eighteen AMR genes were identified, with blaZ (37.5%), qacD (34.7%), and mecA (14.6%) most frequent. Seven isolates carried ≥5 AMR genes. Virulence genes, including cap8E and esxA, were widely distributed. SCCmec elements were identified in all mecA-positive isolates, with type V predominant in ST3 and type II in ST6. Importantly, 76.2% of SCCmec-positive isolates were infection-associated. Novel plasmids carrying multiple resistance genes were identified in two ST3 isolates. This study reveals extensive genetic diversity, widespread AMRs, and virulence determinants in S. lugdunensis, providing insights for surveillance and therapy.IMPORTANCEThis study highlights a direct public health threat: we found that infection-associated strains of S. lugdunensis frequently carry antimicrobial resistance genes and virulence factors, underscoring their potential to cause severe, hard-to-treat infections. It provides a genetic foundation for surveillance: the identification of predominant high-risk lineages and novel plasmids carrying multiple resistance genes offers crucial molecular targets for future tracking and monitoring of this emerging pathogen. It informs clinical decision-making: understanding the genetic basis of its resistance and virulence is a critical step toward developing more effective strategies for infection control and guiding targeted antibiotic therapy.

ABSTRACTPurposeOur study aims to evaluate the characteristics of serum soluble PD‐1 (sPD‐1) and soluble PD‐L1 (sPD‐L1) levels and their correlations with immune status and prognosis in advanced lung cancer patients.MethodsPatients diagnosed with advanced lung cancer based on histology or cytology in Peking University People's Hospital from July 2020 to November 2021 were enrolled. Clinicopathological data were recorded and analyzed. Treatment efficacy was evaluated according to RESIST 1.1 criteria. The serum levels of sPD‐L1 and sPD‐1 were detected by enzyme‐linked immunosorbent assay (ELISA). Lymphocyte subsets were measured by flow cytometry to evaluate the immune status of the patients.ResultsA total of 65 patients with advanced lung cancer were enrolled. sPD‐L1 level in lung cancer patients (15.67 ± 11.09 pg/mL, p = 0.001) was significantly higher than those in healthy controls (5.21 ± 4.46 pg/mL). sPD‐1 level did not show a significant difference between patients with lung cancer and healthy controls. sPD‐L1 level in patients with progressive disease (PD) was significantly higher than those with partial response (PR) (20.94 ± 8.91 vs. 13.14 ± 12.66 pg/mL, p = 0.033). In treatment‐naïve patients, sPD‐L1 level was negatively correlated with the lymphocyte ratio (correlation coefficient = −0.452, p = 0.014). Kaplan–Meier survival analysis showed that patients with low sPD‐L1 level had a significantly longer progression‐free survival (PFS) (10.4 vs. 5.7 months, p = 0.023). However, sPD‐1 level did not correlate with lymphocyte subsets or prognosis in overall patients with lung cancer. Subgroup analysis showed that prolonged PFS in patients with low sPD‐L1 level was exclusively shown in the NSCLC subgroup, not in the SCLC subgroup. In the subgroups of patients who subsequently received immunotherapy, low sPD‐L1 level was correlated with longer PFS in the overall patients and NSCLC patients, and low sPD‐1 level was correlated with longer PFS exclusively in NSCLC patients.ConclusionSerum sPD‐L1 level was higher in patients with advanced lung cancer than healthy individuals, which was negatively correlated with the proportion of lymphocytes and prognosis. Serum sPD‐1 level did not show significant difference between patients with lung cancer and healthy individuals, which showed no correlation with lymphocyte subsets and the prognosis of overall patients, except NSCLC patients receiving immunotherapy.

Background Paclitaxel liposome (Lipusu) is known to be effective in non-small cell lung cancer (NSCLC) as first-line treatment. This study aimed to evaluate the effectiveness and safety of paclitaxel liposome based chemotherapy plus PD-1/PD-L1 inhibitor in patients with advanced NSCLC. Methods In this multicenter, retrospective, real-world study, patients with advanced NSCLC who were administered paclitaxel liposome based chemotherapy plus PD-1/PD-L1 inhibitor in three centers (Peking University People’s Hospital as the lead center) in China between 2016 and 2022 were included. Progression-free survival (PFS), overall survival (OS), objective response rate, disease control rate, and adverse events (AEs) were evaluated. Results A total of 49 patients were included, with 33 (67.3%) receiving paclitaxel liposome based chemotherapy plus PD-1/PD-L1 inhibitor as first-line treatment. There were 34 patients (69.4%) diagnosed with squamous cell carcinoma and 15 (30.6%) with adenocarcinoma. The median follow-up was 20.5 (range: 3.1–41.1) months. The median PFS and OS of all patients were 9.7 months (95% confidence interval [CI], 7.0-12.4) and 30.5 months (95% CI, not evaluable-not evaluable), respectively. Patients with squamous cell carcinoma and adenocarcinoma had median PFS of 11 months (95%CI, 6.5–15.5) and 9.3 months (95%CI, 7.0-12.4), respectively. The median PFS was 9.9 months (95%CI, 7.1–12.7) in patients who received the combined regimen as first-line treatment. Treatment-related AEs of any grade were observed in 25 (51.0%) patients, and AEs of grade 3 or worse were observed in nine patients (18.4%). The most common treatment-related AEs were myelosuppression (14.3%) and fever (10.2%). Conclusions Paclitaxel liposome based chemotherapy plus PD-1/PD-L1 inhibitor prolonged the PFS in advanced NSCLC with acceptable safety, which was worthy of clinical application.

Staphylococcus lugdunensis (S. lugdunensis) is a coagulase-negative Staphylococcus comprising the normal skin microbiota, primarily colonizing the lower abdomen and extremities.[1]S. lugdunensis has attracted substantial attention in recent years since the discovery of lugdunin by Zipperer et al[2] in 2016. Lugdunin is a secondary metabolite synthesized by non-ribosomal peptide synthetase (NRPS) that inhibits the growth of various gram-positive bacteria, including methicillin-resistant S. aureus (MRSA),[2] suggesting its potential as an antibiotic. To date, 28 complete genome sequences and 11 partial-assembly genome sequences of S. lugdunensis have been published and uploaded to the GenBank database (https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/2548/, January 18, 2022). However, there are limited data on the genome sequences of S. lugdunensis from mainland China, and the effects of geographical origin on genetic variations in S. lugdunensis remain unknown. We analyzed six clinical strains of S. lugdunensis isolated from Beijing, Wuhan, and Fujian, China [Supplementary Table 1, https://links.lww.com/CM9/B273]. The minimum inhibitory concentrations (MICs) of antibiotics were evaluated using the VITEK2 Compact system (bioMerieux, Inc., Durham, USA). Cefoxitin disc dilution (cefoxitin DD), broth microdilution for oxacillin, and oxacillin salt agar methods were used as reference methods for assessing methicillin resistance. The complete genomes of the six S. lugdunensis strains were obtained by next-generation sequencing and Oxford Nanopore sequencing, and then compared with genomes available in the GenBank database. We further characterized the resistome and secondary metabolism gene clusters of these staphylococcal cassette chromosome mec (strains using computational approaches. We identified SCCmec) elements of methicillin-resistant S. lugdunensis (MRSL) and performed collinearity analysis of these strains.[3] Comparisons were performed for the obtained nucleotide sequences of the lug operon with those in the GenBank database using Blastx and Blastp tools against the proteins of the operon. We further explored the relationships of genetic variations of the lug operon with geographical origins and the clonal complex (CC). The detailed methods are reported in the Supplementary Materials, https://links.lww.com/CM9/B273. The genome sizes of the six strains ranged from 2.59 to 2.71 Mbp, with a GC content of 33.7% to 33.9% [Supplementary Figure 1, https://links.lww.com/CM9/B273]. All strains contained 2405 to 2523 coding sequences, with 60 to 61 tRNAs, 16 to 19 rRNAs, and 51 to 53 sRNAs. Two to three plasmids were identified in five strains (except RMLUG5). Multilocus sequence typing revealed sequence type (ST)3 (RMLUG1, RMLUG3, RMLUG6), ST27 (RMLUG2), ST34 (RMLUG4), and ST6 (RMLUG5). In antimicrobial susceptibility tests by VITEK2, three of the six strains were resistant to cefoxitin and oxacillin, as confirmed by the oxacillin broth microdilution method and oxacillin salt agar screening. Cefoxitin DD tests showed that strains RMLUG1 and RMLUG6 were susceptible to cefoxitin, although near the breakpoint [Supplementary Table 2, https://links.lww.com/CM9/B273]. The three MRSL strains were susceptible to antibacterial agents showing activity against MRSA, such as linezolid, vancomycin, and teicoplanin [Supplementary Table 3, https://links.lww.com/CM9/B273]). Notably, the methicillin resistance gene mecA was identified in the three MRSL strains (RMLUG1, RMLUG3, and RMLUG6) and in one methicillin-susceptible S. lugdunensis (MSSL) strain (RMLUG2). The SCCmec elements were V(5C2) (RMLUG1, RMLUG2, and RMLUG6) and IVi (2B) (RMLUG3). Only the ccr class 9 (ccrC2 allele 1) gene was identified in the MSSL strain RMLUG4, which is a composite of the SCCmec element. Supplementary Figure 2, https://links.lww.com/CM9/B273 illustrates the SCCmec structure of the six strains. Strains RMLUG1 and RMLUG6 were consistent with SCCmec type V from S. aureus strain WIS [Supplementary Figure 2A, https://links.lww.com/CM9/B273]. Compared with SCCmec type IVi in S. aureus strain JCSC6668, strain RMLUG3 contained a 1014 bp deletion of the gene encoding ISSep1-like transposase [Supplementary Figure 2B, https://links.lww.com/CM9/B273]. The ST27 strain RMLUG2 contained a complete SCCmec type V, including a type mecA class C2 complex and ccrC1 complex, but in a SCCmec type VII-like order: orfX-J3-ccr-J2-mec-J1. Moreover, the IS431 upstream of mecA had the opposite orientation to that downstream, in contrast with the structure of SCCmec type VII, suggesting a novel SCCmec type V variant [Supplementary Figure 2C, https://links.lww.com/CM9/B273]. The SCCmec elements of RMLUG4 did not carry the mecA gene but contained ccr class 9 (ccrC2) and determinants of resistance to heavy metals such as arsenic and copper. According to SCCmec classification,[4] this element is described as an SCC element, which was designated SCCRMLUG4 [Supplementary Figure 2D, https://links.lww.com/CM9/B273]. MIC tests and genomic analysis indicated that strain RMLUG2 carried the mecA gene but did not exhibit oxacillin resistance. No mutation of the mecA gene was observed among the MRSL strains. C to T substitution was identified at position –33 (i.e., 33 bp upstream of the start codon) within the mecA promoter in strain RMLUG2, which also occurred in RMLUG1 and RMLUG6. A comparison of the sequences of FemXAB family genes between strain RMLUG2 and the MRSL strain JICS135 revealed several mutations in femX (eg, R176K and D341E) and femB (eg, D245N and D259Y). The femX and femB genes of strains RMLUG1, RMLUG3, and RMLUG6 matched those of strain JICS135. A comparison of the genomes of the six strains to 14 lug operon genes (lugJ to lugM; Supplementary Table 4, https://links.lww.com/CM9/B273) identified the complete lug operon in all strains except RMLUG2, which only contained lugM. There was a nonsense mutation (g.308T > A) in lugM in strain RMLUG2. Six frameshift mutations were identified in all strains in the genes encoding NRPS enzymes. All frameshift mutations resulted from the deletion of one or two nucleotides, leading to premature termination codons. Additionally, an 18 bp deletion was identified at the beginning of the putative regulator lugR gene in our five strains. An insertion segment, g.67_68insATTTTATACAGGAAGAAG, was identified in lugZ of strains RMLUG4 and RMLUG5. Among all strains, 34 of the 39 genomes in the GenBank database contained 14 complete lug genes, two of which belonged to the same type of strain (NCTC12217) despite different assembly levels from different laboratories. Similar to strain RMLUG2, strains VCU150, VISLISI_25, and C_33 harbored only lugM, but had missense mutations instead of the nonsense mutation. Further analysis of the 38 strains indicated missense mutations in 12 of 14 genes (excluding lugI and lugD), making this the most common mutation type. Missense mutations were detected in lugJ (g.394G > A) in all CC1 isolates. Interestingly, missense mutations were found in lugE (g.331A > G) in 32 of the 38 S. lugdunensis strains, but were not present in any ST1 strain, except strain HKU09-01. Missense mutations in lugG (g.551C > T) were present in all CC6 isolates. In addition, genetic variations in the lug operon were independent of geographical origin. In the current study, the MIC tests and genomic analysis indicated discordance between genotype and phenotype, similar to the findings of Kao et al[5] There are several possible reasons for this discrepancy. First, the heterogeneity of the mecA gene can suppress methicillin resistance. Gargis et al[6] reported that a frameshift mutation in mecA and lack of the full mecA promoter sequence can cause susceptibility to oxacillin and cefoxitin, respectively. Additionally, Chen et al[7] reported that the –33C to T substitution within the mecA promoter can lower the oxacillin MIC, whereas this mutation was identified in strains RMLUG2(MSSL), RMLUG1(MRSL), and RMLUG6(MRSL). Second, mutations in auxiliary genes may contribute to decreased resistance to methicillin. Giannouli et al[8] reported that the accumulation of amino acid changes in FemXAB family proteins may affect cell wall synthesis, leading to atypical oxacillin responsiveness. Several mutations were detected in the femX, femA, and femB genes between strain RMLUG2 and S. lugdunensis JICS135, which might account for the lowered MIC. Moreover, we identified CC-dependent genetic variations of the 14 lug genes, indicating that the lug operon may not be conserved in the S. lugdunensis genome. Lebeurre et al[9] detected significant genetic variations independent of CCs in the lug locus. However, they only searched against genes encoding four NRPS enzymes (lugA, lugB, lugC, and lugD) and one regulator gene (lugR), with the results being somewhat consistent with our findings. Our results indicated that not all S. lugdunensis isolates harbor the lug operon. Four strains containing only lugM varied in clinical source and geographical location; thus, no link was evident between S. lugdunensis lug polymorphisms and geographical origin. There were some limitations to this study. First, we only described the phenotypes and genotypes of the six strains, which could not establish a causal relation. Functional studies are needed to investigate the activity of resistance genes and the lug operon. Second, the GenBank data may be biased because some sequences were obtained only by third-generation sequencing without assembling sequence fragments obtained by other methods. Thus, the accuracy of the sequences should be taken into account. In particular, some nucleotides need to be corrected to confirm the mutations of the lug operon in the future. Overall, our findings revealed variations in the genomes of S. lugdunensis strains isolated from different cities in China. We report novel SCCmec and SCCRMLUG4 elements, suggesting the need for additional studies on this species. Comparative analysis of the lug operon for all available genomes demonstrated CC-dependent genetic variations; however, further research is needed to elucidate the relationships between genotypes and phenotypes. Data availability The complete genome sequences have been deposited at GenBank under the accessions CP084480-CP084483 (RMLUG1), CP084434-CP084436 (RMLUG2), CP084437-CP084439 (RMLUG3), CP084440-CP084442 (RMLUG4), CP084443 (RMLUG5), and CP084444-CP084446 (RMLUG6). Acknowledgments The authors thank Pengcheng Du (Beijing YuanShengKangTai (ProtoDNA) Genetech Co Ltd.) for the technical help in Oxford Nanopore sequencing. We would like to thank Jing Wu for the collection of patient characteristics. We would like to thank Dr. Ronghua Liu (Microbiology Laboratory of Linfen Central Hospital, Linfen, Shaanxi, China) for antibiotic susceptibility test. Funding This work was supported by grants from the National Science and Technology Major Project of China (No. 2017ZX10103004-006) and the National Key Research and Development Programme of China (No. 2016YFC0903800). Conflicts of interest None.

Objective Droplet digital PCR (ddPCR) is a novel assay to detect pneumocystis jjrovecii ( Pj ) which has been defined to be more sensitive than qPCR in recent studies. We aimed to explore whether clinical features of pneumocystis pneumonia (PCP) were associated with ddPCR copy numbers of Pj . Methods A total of 48 PCP patients were retrospectively included. Pj detection was implemented by ddPCR assay within 4 h. Bronchoalveolar fluid (BALF) samples were collected from 48 patients with molecular diagnosis as PCP via metagenomic next generation sequencing (mNGS) or quantitative PCR detection. Univariate and multivariate logistic regression were performed to screen out possible indicators for the severity of PCP. The patients were divided into two groups according to ddPCR copy numbers, and their clinical features were further analyzed. Results Pj loading was a pro rata increase with serum (1,3)-beta-D glucan, D-dimmer, neutrophil percentage, procalcitonin and BALF polymorphonuclear leucocyte percentage, while negative correlation with albumin, PaO2/FiO2, BALF cell count, and BALF lymphocyte percentage. D-dimmer and ddPCR copy number of Pj were independent indicators for moderate/severe PCP patients with PaO2/FiO2 lower than 300. We made a ROC analysis of ddPCR copy number of Pj for PaO2/FiO2 index and grouped the patients according to the cut-off value (2.75). The high copy numbers group was characterized by higher level of inflammatory markers. Compared to low copy number group, there was lower level of the total cell count while higher level of polymorphonuclear leucocyte percentage in BALF in the high copy numbers group. Different from patients with high copy numbers, those with high copy numbers had a tendency to develop more severe complications and required advanced respiratory support. Conclusion The scenarios of patients infected with high ddPCR copy numbers of Pj showed more adverse clinical conditions. Pj loading could reflect the severity of PCP to some extent.

Background Community-acquired pneumonia (CAP) is a major public health challenge worldwide. However, the aetiological and disease severity-related pathogens associated with CAP in adults in China are not well established based on the detection of both viral and bacterial agents. Methods A multicentre, prospective study was conducted involving 10 hospitals located in nine geographical regions in China from 2014 to 2019. Sputum or bronchoalveolar lavage fluid (BALF) samples were collected from each recruited CAP patient. Multiplex real-time PCR and bacteria culture methods were used to detect respiratory pathogens. The association between detected pathogens and CAP severity was evaluated. Results Among the 3,403 recruited eligible patients, 462 (13.58%) had severe CAP, and the in-hospital mortality rate was 1.94% (66/3,403). At least one pathogen was detected in 2,054 (60.36%) patients, with two or more pathogens were co-detected in 725 patients. The ten major pathogens detected were Mycoplasma pneumoniae (11.05%), Haemophilus influenzae (10.67%), Klebsiella pneumoniae (10.43%), influenza A virus (9.49%), human rhinovirus (9.02%), Streptococcus pneumoniae (7.43%), Staphylococcus aureus (4.50%), adenovirus (2.94%), respiratory syncytial viruses (2.35%), and Legionella pneumophila (1.03%), which accounted for 76.06–92.52% of all positive detection results across sampling sites. Klebsiella pneumoniae ( p < 0.001) and influenza viruses ( p = 0.005) were more frequently detected in older patients, whereas Mycoplasma pneumoniae was more frequently detected in younger patients ( p < 0.001). Infections with Klebsiella pneumoniae , Staphylococcus aureus , influenza viruses and respiratory syncytial viruses were risk factors for severe CAP. Conclusions The major respiratory pathogens causing CAP in adults in China were different from those in USA and European countries, which were consistent across different geographical regions over study years. Given the detection rate of pathogens and their association with severe CAP, we propose to include the ten major pathogens as priorities for clinical pathogen screening in China.

Background Community-acquired pneumonia (CAP) is a respiratory disease that frequently requires hospital admission, and is a significant cause of death worldwide. Plasma fetuin-A levels were significantly lower in patients with sepsis, but data regarding CAP are scarce. This study aimed to evaluate the usefulness of fetuin-A as a prognostic biomarker of CAP. Methods A multicenter cohort study on CAP was conducted between January 2017 and December 2018. Demographic and clinical data were recorded for all enrolled patients. Plasma fetuin-A levels were determined using a quantitative enzyme-linked immunosorbent assay. A Cox proportional hazards regression analysis was used to analyse the effect of variables on 30-day mortality. A logistic regression analysis was performed to assess risk factors associated with severe CAP (SCAP) and 30-day mortality. A receiver operating characteristic (ROC) curve was used to verify the association between variables and CAP prognosis. Correlations were assessed using Spearman's test. Survival curves were constructed and compared using the log-rank test. Results A total of 283 patients with CAP were enrolled in this study. Fetuin-A levels were decreased in patients with CAP, especially in SCAP and non-survivors. A cox regression analysis showed that CURB-65 and fetuin-A levels were independent prognostic indicators of 30-day mortality. Via a multiple logistic regression analysis, plasma level of fetuin-A (<202.86 mg/L) was determined to be the strongest independent predictor of 30-day mortality considered (odds ratio, 57.365), and also was also determined to be an independent predictor of SCAP. The area under the curve (AUC) of fetuin-A for predicting 30-day mortality was 0.871, and accuracy was high ( P < 0.05). Plasma fetuin-A levels were negatively correlated with WBC, NE%, Glu, CRP, PCT, CURB-65, and pneumonia severity index scores and positively correlated with albumin level. Kaplan–Meier curves showed that lower plasma levels of fetuin-A levels were associated with increased 30-day mortality levels ( P < 0.0001). Conclusion Plasma fetuin-A levels were decreased in patients with CAP. Fetuin-A can reliably predict mortality in patients with CAP, and is a useful diagnostic indicator of SCAP.

Background Metagenomic next-generation sequencing (mNGS) is an important supplement to conventional tests for pathogen detections of pneumonia. However, mNGS pipelines were limited by irregularities, high proportion of host nucleic acids, and lack of RNA virus detection. Thus, a regulated pipeline based on mNGS for DNA and RNA pathogen detection of pneumonia is essential. Methods We performed a retrospective study of 151 patients with pneumonia. Three conventional tests, culture, loop-mediated isothermal amplification (LAMP) and viral quantitative real-time polymerase chain reaction (qPCR) were conducted according to clinical needs, and all samples were detected using our optimized pipeline based on the mNGS (DNA and RNA) method. The performances of mNGS and three other tests were compared. Human DNA depletion was achieved respectively by MolYsis kit and pre-treatment using saponin and Turbo DNase. Three RNA library preparation methods were used to compare the detection performance of RNA viruses. Results An optimized mNGS workflow was built, which had only 1-working-day turnaround time. The proportion of host DNA in the pre-treated samples decreased from 99 to 90% and microbiome reads achieved an approximately 20-fold enrichment compared with those without host removal. Meanwhile, saponin and Turbo DNase pre-treatment exhibited an advantage for DNA virus detection compared with MolYsis. Besides, our in-house RNA library preparation procedure showed a more robust RNA virus detection ability. Combining three conventional methods, 76 (76/151, 50.3%) cases had no clear causative pathogen, but 24 probable pathogens were successfully detected in 31 (31/76 = 40.8%) unclear cases using mNGS. The agreement of the mNGS with the culture, LAMP, and viral qPCR was 60%, 82%, and 80%, respectively. Compared with all conventional tests, mNGS had a sensitivity of 70.4%, a specificity of 72.7%, and an overall agreement of 71.5%. Conclusions A complete and effective mNGS workflow was built to provide timely DNA and RNA pathogen detection for pneumonia, which could effectively remove the host sequence, had a higher microbial detection rate and a broader spectrum of pathogens (especially for viruses and some pathogens that are difficult to culture). Despite the advantages, there are many challenges in the clinical application of mNGS, and the mNGS report should be interpreted with caution.

BackgroundPneumonia is a leading cause of non-relapse mortality after hematopoietic stem cell transplantation (HSCT), and the lower respiratory tract (LRT) microbiome has been proven to be associated with various respiratory diseases. However, little is known about the characteristics of the LRT microbiome in patients with post-HSCT compared to healthy controls (HC) and community-acquired pneumonia (CAP).MethodsBronchoalveolar lavage samples from 55 patients with post-HSCT pneumonia, 44 patients with CAP, and 30 healthy volunteers were used to detect microbiota using 16S rRNA gene sequencing.ResultsThe diversity of the LRT microbiome significantly decreased in patients with post-HSCT pneumonia, and the overall community was different from the CAP and HC groups. At the phylum level, post-HSCT pneumonia samples had a high abundance of Actinobacteria and a relatively low abundance of Bacteroidetes. The same is true for non-survivors compared with survivors in patients with post-HSCT pneumonia. At the genus level, the abundances of Pseudomonas, Acinetobacter, Burkholderia, and Mycobacterium were prominent in the pneumonia group after HSCT. On the other hand, gut-associated bacteria, Enterococcus were more abundant in the non-survivors. Some pathways concerning amino acid and lipid metabolism were predicted to be altered in patients with post-HSCT pneumonia.ConclusionsOur results reveal that the LRT microbiome in patients with post-HSCT pneumonia differs from CAP patients and healthy controls, which could be associated with the outcome. The LRT microbiota could be a target for intervention during post-HSCT pneumonia.

Abstract Background A novel coronavirus (SARS-CoV-2) has infected more than 75,000 individuals and spread to over 20 countries. It is still unclear how fast the virus evolved and how the virus interacts with other microorganisms in the lung. Methods We have conducted metatranscriptome sequencing for the bronchoalveolar lavage fluid of eight SARS-CoV-2 patients, 25 community-acquired pneumonia (CAP) patients, and 20 healthy controls. Results The median number of intra-host variants was 1-4 in SARS-CoV-2 infected patients, which ranged between 0 and 51 in different samples. The distribution of variants on genes was similar to those observed in the population data (110 sequences). However, very few intra-host variants were observed in the population as polymorphism, implying either a bottleneck or purifying selection involved in the transmission of the virus, or a consequence of the limited diversity represented in the current polymorphism data. Although current evidence did not support the transmission of intra-host variants in a person-to-person spread, the risk should not be overlooked. The microbiota in SARS-CoV-2 infected patients was similar to those in CAP, either dominated by the pathogens or with elevated levels of oral and upper respiratory commensal bacteria. Conclusion SARS-CoV-2 evolves in vivo after infection, which may affect its virulence, infectivity, and transmissibility. Although how the intra-host variant spreads in the population is still elusive, it is necessary to strengthen the surveillance of the viral evolution in the population and associated clinical changes.

The outbreaks of 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 infection have posed a severe threat to global public health. It is unclear how the human immune system responds to this infection. Here, we used metatranscriptomic sequencing to profile immune signatures in the bronchoalveolar lavage fluid of eight COVID-19 cases. The expression of proinflammatory genes, especially chemokines, was markedly elevated in COVID-19 cases compared to community-acquired pneumonia patients and healthy controls, suggesting that SARS-CoV-2 infection causes hypercytokinemia. Compared to SARS-CoV, which is thought to induce inadequate interferon (IFN) responses, SARS-CoV-2 robustly triggered expression of numerous IFN-stimulated genes (ISGs). These ISGs exhibit immunopathogenic potential, with overrepresentation of genes involved in inflammation. The transcriptome data was also used to estimate immune cell populations, revealing increases in activated dendritic cells and neutrophils. Collectively, these host responses to SARS-CoV-2 infection could further our understanding of disease pathogenesis and point toward antiviral strategies.

结缔组织病(CTD)是一组侵犯全身结缔组织的多系统疾病,感染是结缔组织病患者的重要死因之一[1,2].结缔组织病常累及呼吸系统,患者同时伴有免疫紊乱,易并发肺部感染,糖皮质激素和免疫抑制药物的应用增加了这类患者发生重症肺炎的可能性[3].本研究对北京大学人民医院呼吸科普通病房及重症监护病房(ICU)收治的CTD免疫抑制治疗后并发肺炎29例进行总结分析,旨在为临床诊治提供参考.

An outbreak of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that started in Wuhan, China, at the end of 2019 has become a global pandemic. Both SARS-CoV-2 and SARS-CoV enter host cells via the angiotensin-converting enzyme 2 (ACE2) receptor, which is expressed in various human organs. We have reviewed previously published studies on SARS and recent studies on SARS-CoV-2 infection, named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO), confirming that many other organs besides the lungs are vulnerable to the virus. ACE2 catalyzes angiotensin II conversion to angiotensin-(1–7), and the ACE2/angiotensin-(1–7)/MAS axis counteracts the negative effects of the renin-angiotensin system (RAS), which plays important roles in maintaining the physiological and pathophysiological balance of the body. In addition to the direct viral effects and inflammatory and immune factors associated with COVID-19 pathogenesis, ACE2 downregulation and the imbalance between the RAS and ACE2/angiotensin-(1–7)/MAS after infection may also contribute to multiple organ injury in COVID-19. The SARS-CoV-2 spike glycoprotein, which binds to ACE2, is a potential target for developing specific drugs, antibodies, and vaccines. Restoring the balance between the RAS and ACE2/angiotensin-(1–7)/MAS may help attenuate organ injuries. Graphical abstract SARS-CoV-2 enters lung cells via the ACE2 receptor. The cell-free and macrophage-phagocytosed virus can spread to other organs and infect ACE2-expressing cells at local sites, causing multi-organ injury.

Background. A novel coronavirus (CoV), severe acute respiratory syndrome (SARS)-CoV-2, has infected >75 000 individuals and spread to >20 countries. It is still unclear how fast the virus evolved and how it interacts with other microorganisms in the lung. Methods. We have conducted metatranscriptome sequencing for bronchoalveolar lavage fluid samples from 8 patients with SARS-CoV-2, and also analyzed data from 25 patients with community-acquired pneumonia (CAP), and 20 healthy controls for comparison. Results. The median number of intrahost variants was 1-4 in SARS-CoV-2-infected patients, ranged from 0 to 51 in different samples. The distribution of variants on genes was similar to those observed in the population data. However, very few intrahost variants were observed in the population as polymorphisms, implying either a bottleneck or purifying selection involved in the transmission of the virus, or a consequence of the limited diversity represented in the current polymorphism data. Although current evidence did not support the transmission of intrahost variants in a possible person-to-person spread, the risk should not be overlooked. Microbiotas in SARS-CoV-2-infected patients were similar to those in CAP, either dominated by the pathogens or with elevated levels of oral and upper respiratory commensal bacteria. Conclusion. SARS-CoV-2 evolves in vivo after infection, which may affect its virulence, infectivity, and transmissibility. Although how the intrahost variant spreads in the population is still elusive, it is necessary to strengthen the surveillance of the viral evolution in the population and associated clinical changes.

The outbreaks of 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 infection has posed a severe threat to global public health. It is unclear how the human immune system responds to the virus infection. Here, we profiled the immune transcriptome signatures by metatranscriptome sequencing for the bronchoalveolar lavage fluid from eight COVID-19 cases. The expression of the proinflammatory genes, especially chemokines, was markedly elevated in COVID-19 cases as compared to community-acquired pneumonia patients and healthy controls, suggesting that SARS-CoV-2 infection caused hypercytokinemia. Contrasting with SARS-CoV, which is thought to induce inadequate interferon (IFN) response, SARS-CoV-2 robustly triggered the expression of myriad IFN-inducible genes (ISGs). These ISGs exhibit immunopathogenic potentials, characterized by the overrepresentation of genes involved in inflammation. Collectively, we profiled the molecular signatures of the host response to SARS-CoV-2 infection, which could help to understand the disease pathogenesis and provided clues for tailored antiviral strategies, such as IFN therapy.

Background: The value of procalcitonin (PCT) in the diagnosis of bacterial infections and for determining antibiotic usage among patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) is currently unclear. Methods: We systematically reviewed the literature and selected studies that evaluated PCT as a biomarker for predicting bacterial infection and compared PCT-based protocols to determine its application in the initiation or discontinuation of antibiotics. Guidance for systematic reviews from Cochrane and the GRADE were followed to perform this study. Data were pooled and analyzed by using a random-effects or a fixed-effects model based on the heterogeneity. Results: The pooled sensitivity and specificity of PCT in diagnosing respiratory bacterial infections were 0.60 and 0.76, respectively, with the area under the summary receiver operating characteristic curve of 0.77. Subgroup analysis showed that the sensitivity and specificity of PCT for patients in ICU were 0.48 and 0.69, respectively. PCT-based protocols decreased antibiotic prescription (relative risk = 0.66, 95% CI: 0.62-0.71) and total antibiotic exposure (mean difference = -2.60, 95% CI: -4.48-0.72), without affecting clinical outcomes such as treatment failure, length of hospitalization and rates of re-exacerbation or overall mortality. Conclusions: PCT has a moderate ability to distinguish bacterial respiratory infection in patients with AECOPD. PCT-guided algorithm can reduce unnecessary administration of antibiotics without increasing adverse outcomes. However, for patients requiring admission in the ICU, PCT may have a poor diagnostic value, and the PCT-guided algorithm may not effectively and safely reduce the antibiotic exposure.

Purpose: Prognostic biomarkers help triage initial patients and inform targeted therapy selection. Here, we explored the role of progranulin (PGRN)-implicated in processes ranging from inflammation to neurodegeneration-in patients with community-acquired pneumonia (CAP). Methods: A prospective observational cohort study was conducted during 2017. Patients who required invasive mechanical ventilation and/or had septic shock and were discharged from the hospital were cohort II. Those who died at the hospital were cohort III. Remaining patients discharged from the hospital were cohort I. The primary endpoint was that patients progressed to served as cohort II; the secondary endpoint was that patients progressed to served as cohort III. Serum PGRN levels were detected by ELISA. Results: A total of 280 patients constituted the study cohort. 194 (69.3%) were categorized into cohort I, 61 (21.8%) were categorized into cohort II, and 25 (8.9%) were categorized into cohort III. Serum PGRN levels were increased in CAP patients, independently of etiology. Adjusting for clinical parameters, the odds ratios (95%CI) of cohort III and combined cohort II-III were 34.968 (3.743-326.692) and 3.741 (1.496-9.351), respectively, comparing lowest-to-highest quartile PGRN levels. PGRN exhibited high accuracy in predicting 30-day mortality, with AUC 0.862. PGRN combined with CURB-65 or PSI significantly improved prediction performance. Cox proportional regression analysis showed PGRN was an independent predictor for 30-day mortality risk. Cox survival curves confirmed PGRN >= 89.51 ng/mL had a significantly higher mortality rate than PGRN <89.51 ng/mL. Conclusion: Higher PGRN levels at admission were associated with higher odds of poor prognosis. PGRN can improve the prognostic power of CURB-65 or PSI, so PGRN could be apparently a prognostic biomarker for assisting triage of CAP patients. (C) 2019 The British Infection Association. Published by Elsevier Ltd. All rights reserved.