Objective:To analyze the morphology and molecular biology of Rhodococcus erythropolis isolated from blood culture, and clarify its microbiological characteristics, clinical diagnosis and treatment. Methods:Strain F1069 was isolated and cultured. Then, it was analyzed by morphology, physiological tests, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS), 16S rRNA gene sequencing, and whole-genome sequencing analysis.Results:The colonies of Rhodococcus erythropolis were light yellow, moist, round, and raised, and had neatly-edged margins after being cultured for 48 h. They could turn orange-red after a prolonged cultivation time. The strain was gram-positive bacillus without spores and was negative in acid-fast staining. The strain was identified as Rhodococcus erythropolis by MALDI-TOF MS, and the result was confirmed by 16S rRNA gene sequencing. The F1069 strain contained the RbpA resistance gene and multiple virulence genes. Conclusions:Cases of Rhodococcus erythropolis infection are rare. The diagnosis of such cases depends on the pathogen detection results, especially molecular biology methods. A definitive diagnosis enables rapid guidance for clinical anti-infection treatment.

Cytokine storm is a common and fatal pathological process in patients with severe infections. Its core mechanism involves the excessive activation of the immune system, leading to a massive release of cytokines, which in turn causes acute inflammatory responses and multiple organ failure. Studying the mechanisms and treatment methods of cytokine storm is of great significance for improving the survival rate of patients with severe infections. This paper systematically elucidates the pathogenesis, pathological characteristics, and current progress in the treatment of cytokine storm in severe infections, discusses the treatment strategies such as anti-inflammatory drugs, immunomodulatory therapy, and supportive care, and summarizes the results of clinical trials of emerging drugs and methods for treating cytokine storm, hoping to provide theoretical evidence and practical guidance for the precise diagnosis and treatment of cytokine storm and provide reference for clinical treatment of patients with severe infections.

Objective:To establish and verify an indirect ELISA for quantitative detection of serum antibody against varicella-zoster virus (VZV).Methods:Recombinant VZV glycoprotein E (gE) was diluted with the coating buffer to concentrations of 2, 0.5, and 0.25 μg/ml. HRP-labeled antibody dilutions were 1∶2 000, 1∶5 000, and 1∶10 000. The optimal coating concentration and the optimal working concentration were determined according to the A450/630 value and the P/N ratio (working reference A450/630 value/blank control A450/630 value). According to the indirect ELISA process, an indirect ELISA quantitative detection method for VZV-specific antibody was established. The linear range, accuracy, and precision of the method were verified. The titers of VZV-specific IgG antibody in 32 serum samples were detected by the established method, fluorescent antibody to membrane antigen (FAMA) test, and commercially available VZV quantitative ELISA kit, and the results of the three methods were compared. Results:The optimal coating concentration was 0.25 μg/ml and the optimal working concentration of HRP-labeled antibody was 1∶10 000. The linear range of the standard curve was 0.015 7-1 mIU/ml; the recovery rate was 95.32%-110.99%, and the coefficient of variation (CV) values for repeatability and the intermediate precision were both less than 10%. The results of the established indirect ELISA were well correlated with the results of FAMA test and commercial ELISA kit (indirect ELISA & FAMA: the correlation coefficient was 0.886 4, indirect ELISA & commercially ELISA kit: the correlation coefficient was 0.806 6).Conclusions:The established indirect ELISA method for serum anti-VZV antibody detection has good accuracy and precision, and shows a good correlation with FAMA test and commercial VZV ELISA kit.

Objective:To investigate the role of the structural gene vgrG of the type Ⅵ secretion system (T6SS) of Klebsiella pneumoniae ( Kpn), and evaluate the growth ability in vitro and pathogenicity of the bacteria after vgrG was deleted. Methods:Using sequences published by the National Center for Biotechnology Information (NCBI), primers were designed to amplify the upstream and downstream homology arms of vgrG via PCR. These fragments were cloned into the vector pKO3-Km after overlapping, the recombinant vector pKO3-Km- vgrG was transferred into Kpn competent cells, and the vgrG deletion strain Δ vgrG was obtained through homologous recombination. The vgrG promoter with the complete gene fragment was amplify by PCR and cloned into the pBAD33 vector. The pBAD33- vgrG was then transferred into Δ vgrG competent cells to obtain the complemented strain CΔ vgrG. The wild-type strain (WT), Δ vgrG strain and CΔ vgrG strain were cultured in LB (Luria-Bertani) liquid medium to compare growth rates. Adhesion to human lung epithelial A549 cells and intracellular survival in macrophages Raw264.7 cells were assessed. In vivo experiments included mouse survival analysis ( n=10) and lung bacterial load quantification ( n=6). Statistical comparisons were performed using the Student t-test. Results:The Δ vgrG strain was obtained through homologous recombination. It was identified by specific primers that compared with the WT strain, the complete vgrG fragment (2 487 bp) was deleted. On this basis, the CΔ vgrG strain was obtained. Deletion of vgrG did not significantly affect Kpn growth in vitro growth ability of bacteria before on after Kpn deleted vgrG [(1.40±0.10) vs (1.20±0.30), t=0.63, P>0.05]. The viscosity of WT strain was significantly higher than that of the Δ vgrG strain [(0.96±0.04) vs (0.38±0.05), t=9.72, P<0.05], the viscosity of the CΔ vgrG strain was also significantly higher than that of the Δ vgrG strain ( P<0.05). At the cellular level, the amount of adherent bacteria of the WT strain to A549 cells was significantly greater than that of the Δ vgrG strain [(5 367.00±318.00) CFU vs (4 067.00±88.19) CFU, t=3.94, P<0.05], the amount of adherent bacteria of CΔ vgrG strain was also significantly higher than that of Δ vgrG strain ( P<0.05). After 12 h infection, the WT strain survival rate in macrophages was significantly higher than that of the Δ vgrG strain[(69.00±1.00)% vs (47.50±2.50)%, t=7.99, P<0.05]. At the animal level, the survival rate of WT strain group after lethal dose infection of mice was significantly lower than that of Δ vgrG strain group [(16.67±8.82)% vs (53.33±6.67)%, t=3.32, P<0.05]; mice infected with semi-lethal dose and the number of bacteria load in the lungs of WT strain group was significantly higher than that of the Δ vgrG strain group[ (4.97±0.06) lg CFU/g vs (4.05 ±0.04) lg CFU/g, t=12.27, P<0.01], the amount of bacteria in the lungs of mice in CΔ vgrG strain group was also significantly higher than that in Δ vgrG strain group ( P<0.01). Conclusions:The vgrG gene does not affect the growth of Kpn in vitro, but it is involved in the adhesion of Kpn to epithelial cells, resistance to macrophage killing and pathogenicity to mice.

Objective:To analyze the distribution and epidemiological characteristics of non-bacterial pathogens in hospitalized children with acute respiratory infections at a tertiary pediatric hospital in Shanghai during 2023.Methods:A retrospective study was conducted on 10 591 children with acute respiratory tract infections who were hospitalized in Shanghai Children's Hospital from January to December 2023. A multiplex PCR combined with capillary electrophoresis platform was used to detect 11 common non-bacterial respiratory pathogens（including viruses and atypical pathogens）. Statistical analysis was carried out using SPSS 29.0 software. Qualitative data were presented as numbers and percentages，and the Chi-square test was employed to make comparisons between groups，aiming to analyze the differences in the distribution of different pathogens according to gender，age group，and season. Additionally，based on the severity of the disease，patients were calssified into a severe pneumonia group and a non-severe pneumonia group to further explore the characteristics of the pathogen spectrum of severe pneumonia.Results:The total detection rate of pathogens was 54.39%（5 760/10 591），and the proportion of mixed infections was 12.76%（735/5 760）. The dominant pathogens and their proportions were as follows： Mycoplasma pneumoniae（19.20%，2 034/10 591），human rhinovirus（12.16%，1 288/10 591），influenza A virus（8.31%，880/10 591），and respiratory syncytial virus（8.14%，862/10 591）. Epidemiological characteristics showed that：（1）In terms of age： Mycoplasma pneumoniae was more common in older children（29.55%，901/3 049，in the school-age group，χ 2 = 653.67， P<0.001）. Influenza A virus had a high incidence in the adolescent group（11.34%，45/397，χ 2=48.69， P<0.001）. Respiratory syncytial virus was most susceptible in the infant group（20.94%，280/1 337，χ 2=739.92， P<0.001）. Human rhinovirus showed the characteristic of general susceptibility across all ages.（2）Monthly and seasonal distribution： Mycoplasma pneumoniae had a seasonal epidemic in summer and autumn（it began to rise in May and peaked in October at 34.22%，439/1 283）；influenza A virus had a bimodal distribution in spring and winter（the peak was 37.15% in March，315/848）；respiratory syncytial virus had a dominant epidemic in spring and summer（the detection rate was 21.24% in May，206/970），and human rhinovirus was prevalent throughout the year.（3）Clinical correlation：The detection rate of pathogens in the severe pneumonia group was significantly higher than that in the non-severe group：84.19%（426/506） vs 2.89%（5 334/10 085），χ 2=56.23， P<0.001. Conclusions:In 2023，the pathogen spectrum of hospitalized children with acute respiratory infections in the Shanghai area exhibits an epidemic pattern dominated by Mycoplasma pneumoniae，and its transmission dynamics are significantly age-dependent. This study delineates the pathogen-host-environment tripartite interactions，establishing an evidence-based foundation for formulating precision diagnostic-therapeutic algorithms and seasonal nosocomial infection prevention frameworks.

T cell factor 1（TCF-1）is a downstream transcription factor of the Wnt/β-catenin signaling pathway，and plays an important role in the development，differentiation，and memory formation of T cells. Recent studies have shown that TCF-1 can regulate the formation and maintenance of stem-like memory T cells（Tscm），and has potential application value in evaluating the prognosis of tumor immunotherapy and as a target for tumor immunotherapy. This article reviews the regulatory effects of TCF-1 on the immune memory as well as stemness formation and maintenance of CD8 +T cells，summarizes the transcription network centered on TCF-1，and further elucidates the application and value of TCF-1 in tumor immunotherapy.

Objective:To investigate the expression of family with sequence similarity 110 member B (FAM110B) and analyze its associations with clinical prognosis, tumor heterogeneity, and immune-related gene expression through a pan-cancer analysis.Methods:TCGA and GTEx databases were used to evaluate the differential expression of FAM110B at mRNA level in pan-cancer and normal tissues. SangerBox platform and TISIDB database were used to analyze the associations of the expression level of FAM110B mRNA with clinical stages, histological grades, and overall survival of patients. The cBioPortal database and the GSCALite platform were used to analyze the genetic variations in the FAM110B gene, and the associations of FAM110B expression with immune regulatory genes, immune checkpoints, tumor mutation burden, microsatellite instability, and anti-cancer drug sensitivity. qRT-qPCR and Western blot were used to detect the expression of FAM110B at mRNA and protein levels in pancreatic cancer, respectively. Independent samples t-test was employed to assess the significance of differences between two groups; Spearman correlation coefficient was used to evaluate the associations between variables; Log-rank test was used for survival analysis. Results:FAM110B was abnormally expressed in a variety of tumors and associated with the overall survival of patients ( P<0.05), with the most significant difference observed in pancreatic cancer ( P<0.001). In vitro experiments verified that FAM110B was highly expressed in PANC-1 cells ( P<0.01), a pancreatic cancer cell line, and its expression level was related to pathological staging and histological grading ( P<0.001). In addition, the expression level of FAM110B mRNA was related to the expression levels of multiple immunomodulatory genes and correlated with tumor mutational burden and microsatellite instability in various tumors ( P<0.05). Conclusions:FAM110B is related to the prognosis, immune regulation, and tumor heterogeneity across multiple cancers, demonstrating promising potential in both basic research and clinical treatment of various cancers.

Objective:To investigate the clinical data, drug resistance and treatment prognosis of Cryptococcus neoformans isolated from patients with acquired immunodeficiency syndrome(AIDS) in a hospital in Shanghai. Methods:The clinical data of AIDS patients with Cryptococcus neoformans infection in Shanghai Public Health Clinical Center from January 2014 to December 2023, and the drug sensitivity to 5 antifungal drugs in vitro, treatment and prognosis were retrospectively analyzed. Results:From January 2014 to December 2023, there were 295 AIDS patients with Cryptococcus neoformans infection in our hospital, with 255 males and 40 females. CD4 + T lymphocyte counts ≤100 cells/μl were detected in 251 patients. A total of 384 strains of Cryptococcus neoformans were isolated from the 295 patients, with the highest detection rate in cerebrospinal fluid samples (65.9%, 253/384), followed by blood samples (29.4%, 113/384). The sensitivity of 384 strains of Cryptococcus neoformans to 5-fluorocytosine was the highest (98.5%, 379/384), followed by fluconazole (95.6%, 367/384) and amphotericin B (95.3%, 366/384). After treatment against cryptococcal infection, 252 patients (86.0%, 252/293) were discharged and 20 patients (6.8%, 20/293) died. The other 2 cases were not treated for cryptococcal infection. Conclusions:As Cryptococcus neoformans is an important pathogen of AIDS patients, clinicians should actively carry out laboratory examination of Cryptococcus and rational drug use according to the results of drug sensitivity test, while alert to the occurrence of drug resistance.

Objective:To identify and trace the origin of the Francisella tularensis subsp. novicida strain SJCS-979 isolated from the blood of a patient, so as to provide a reference for the traceability investigation of such infection events. Methods:Hot spring water samples that the patient had recently bathed in were collected to culture the causative agent, combined with the pathogenic characteristics and the patient′s activity before the bloodstream infection. The water samples were concentrated, acid-treated, and then the isolation of the causative agent was performed, following the method for Legionella detection in circulating cooling water. Suspected strains detected from the hot spring water were subjected to classical phenotypic identification, API ZYM and API NH strips tests, drug sensitivity testing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification, and the obtained data were compared with those of strain SJCS-979 isolated from the patient′s blood. A phylogenetic tree was constructed based on genomic analysis to determine the taxonomic status of strain SJCS-979 and related strains. Epidemiological data of Francisella tularensis subsp. novicida were collected and analyzed by integrating the global genetic and genomic data on GenBank database. Core single nucleotide polymorphism(SNP) comparisons were obtained using Snippy 3.2 software, and then an evolutionary tree was built to determine its population structure based on BAPS analysis. Results:Strain CC-2, isolated from the hot spring water, shared the same biochemical and drug sensitivity phenotype, and had a nearly consistent mass spectrometric profile with strain SJCS-979 isolated from the blood of a patient. Genomic phylogenetic tree analysis based on 120 core protein sequences showed that strains SJCS-979 and CC-2 fell on the same branch with known Francisella tularensis subsp. novicida strains. Bayesian genotyping showed that the global Francisella tularensis subsp. novicida strains with genomic data could be divided into six different sequence clusters. Strains SJCS-979 and CC-2 were located in the same taxonomic group with only 4 SNP differences, indicating that they might be the same clone. Conclusions:This study reports a case of bacteremia caused by Francisella tularensis subsp. novicida, and natural hot spring water may be the environmental source of this infection event.

Objective:To construct a VP8-mRNA vaccine using human rotavirus spike protein VP8 domain as the immunogen and analyze its immunogenicity in mice.Methods:The VP8-mRNA sequence was designed, optimized, and synthesized. The VP8 gene of rotavirus G1P[8] type was used to construct the plasmid pUC57-VP8-Kan-SapⅠ, which was then sequenced. The plasmid confirmed by sequencing was subjected to large-scale amplification and extraction, followed by linearization, in vitro transcription, and capping. The purified capped products were encapsulated with lipid nanoparticles using a microfluidic control apparatus. The encapsulated VP8-mRNA vaccine was administered intramuscularly to mice at 10, 15, and 20 μg. Serum samples were collected for antibody detection by ELISA. Cellular immune responses were detected by flow cytometry and ELISPOT. Statistical analysis was performed using one-way or two-way analysis of variance and Tukey-Kramer test. Results:The encapsulated VP8-mRNA vaccine was rounded and spherical, with a particle size of about 100 nm, a polymer dispersion index of 0.088, and an encapsulation rate of 92.3%. Two doses of VP8-mRNA vaccine immunization could induce a good immune response in mice. The level of IgG antibody induced after immunization in the 15 μg group was comparable to that of the 20 μg group, and there was no statistical difference ( P>0.05), but the antibody levels in the two groups were significantly higher than that in the 10 μg group ( P<0.000 1). VP8-mRNA vaccine could induce neutralizing antibodies against rotavirus G1 and G9 types. The highest level of neutralizing antibodies against rotavirus type G1 was observed in the 15 μg group, which was significantly higher than that in the 10 μg group ( P<0.05). All immunization groups exhibited good neutralizing ability against rotavirus G9 type. The results of ELISPOT showed that lymphocytes from mice in each vaccine group were able to secrete IFN-γ when stimulated with VP8 peptide. Flow cytometry showed that the proportions of CD8 + T cell subsets in the vaccine groups were higher than that in the control group. Conclusion:The VP8-mRNA vaccine has good immunogenicity in mice and can induce good humoral and T-cell immune responses.