To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

This study was aimed at analyzing the epidemiological and drug resistance characteristics of tuberculosis at a desig-nated tuberculosis hospital in Hunan Province in 2024.Patients diagnosed with TB at the hospital between April and October 2024 were included in the study.Demographic data,clinical information,and drug sensitivity test results were collected from the hospital′s electronic medical record system.Descriptive statistics,the chi-square test,and logistic regression were used to analyze the epidemic characteristics,drug resistance characteristics,and factors influencing tuberculosis.Whole genome sequencing of isolates was per-formed,and lineage classification and drug resistance gene mutations were detected with TB-Profiler.The male-to-female ratio was 2.72∶1,and the median age was 56(IQR:43-66)years.Among the 391 patients,most were farmers(46.8%,183/391)and were pri-marily from Changsha(41.1%,162/391).Significant differences were observed in sex and occupation between pulmonary tuberculosis(PTB)and extrapulmonary tuberculosis(EPTB).The overall prevalence of any type of drug resistance of tuberculosis was 33.25%,and the multidrug resistance TB(MDR-TB)and poly-drug resistance(PR-TB)rates were 14.23%and 4.35%,respectively.The re-sistance rates to rifampicin(RIF),isoniazid(INH),ethambutol(EMB),and streptomycin(SM)were 17.90%,22.25%,6.39%,and 20.20%,respectively.Multivariable logistic regression analysis indicated that both diabetes(OR:2.295,95%CI:1.082-4.866)and retreatment(OR:17.822,95%CI:8.343-38.072)were risk factors for developing MDR-TB.Lineage 2(L2)strains accounted for 64.40%(136/191),whereas lineage 4(L4)accounted for 28.80%(55/191).The most common drug resistance mutations were katG Ser315Thr(62.50%,20/32)for INH,rpoB Ser450Leu(50.00%,12/24)for RIF,embB Met306Val(55.56%,5/9)for EMB,and rpsL Lys43Arg(80.95%,34/42)for SM.In conclusion,TB drug resistance was found to be a serious problem at a designated tu-berculosis hospital in Hunan in 2024.Strengthening the treatment and management of patients infected with L2 strains,those with co-morbid diabetes,and retreatment cases is crucial for preventing and controlling the emergence of drug-resistant TB.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

This study was aimed at analyzing the epidemiological and drug resistance characteristics of tuberculosis at a desig-nated tuberculosis hospital in Hunan Province in 2024.Patients diagnosed with TB at the hospital between April and October 2024 were included in the study.Demographic data,clinical information,and drug sensitivity test results were collected from the hospital′s electronic medical record system.Descriptive statistics,the chi-square test,and logistic regression were used to analyze the epidemic characteristics,drug resistance characteristics,and factors influencing tuberculosis.Whole genome sequencing of isolates was per-formed,and lineage classification and drug resistance gene mutations were detected with TB-Profiler.The male-to-female ratio was 2.72∶1,and the median age was 56(IQR:43-66)years.Among the 391 patients,most were farmers(46.8%,183/391)and were pri-marily from Changsha(41.1%,162/391).Significant differences were observed in sex and occupation between pulmonary tuberculosis(PTB)and extrapulmonary tuberculosis(EPTB).The overall prevalence of any type of drug resistance of tuberculosis was 33.25%,and the multidrug resistance TB(MDR-TB)and poly-drug resistance(PR-TB)rates were 14.23%and 4.35%,respectively.The re-sistance rates to rifampicin(RIF),isoniazid(INH),ethambutol(EMB),and streptomycin(SM)were 17.90%,22.25%,6.39%,and 20.20%,respectively.Multivariable logistic regression analysis indicated that both diabetes(OR:2.295,95%CI:1.082-4.866)and retreatment(OR:17.822,95%CI:8.343-38.072)were risk factors for developing MDR-TB.Lineage 2(L2)strains accounted for 64.40%(136/191),whereas lineage 4(L4)accounted for 28.80%(55/191).The most common drug resistance mutations were katG Ser315Thr(62.50%,20/32)for INH,rpoB Ser450Leu(50.00%,12/24)for RIF,embB Met306Val(55.56%,5/9)for EMB,and rpsL Lys43Arg(80.95%,34/42)for SM.In conclusion,TB drug resistance was found to be a serious problem at a designated tu-berculosis hospital in Hunan in 2024.Strengthening the treatment and management of patients infected with L2 strains,those with co-morbid diabetes,and retreatment cases is crucial for preventing and controlling the emergence of drug-resistant TB.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Objective    To evaluate the diagnostic value of various severity assessment scoring systems for sepsis after cardiac surgery and the predictive value for long-term prognosis. Methods    The clinical data of patients who underwent cardiac sugeries including coronary artery bypass grafting (CABG) and (or) valve reconstruction/valve replacement were extracted from Medical Information Mark for Intensive Care-Ⅲ (MIMIC-Ⅲ). A total of 6 638 patients were enrolled in this study, including 4 558 males and 2 080 females, with an average age of 67.0±12.2 years. Discriminatory power was determined by comparing the area under the receiver operating characteristic (ROC) curve (AUC) for each scoring system individually using the method of DeLong. An X-tile analysis was used to determine the optimal cut-off point for each scoring system, and the patients were grouped by the cut-off point, and Kaplan-Meier curves and log-rank test were applied to analyze their long-term survival. Results    Compared with the sequential organ failure assessment (SOFA) score, acute physiology score-Ⅲ (APS-Ⅲ, P<0.001), the simplified acute physiology score-Ⅱ (SAPS-Ⅱ, P<0.001) and logistic organ dysfunction score (LODS, P<0.001) were more accurate in distinguishing sepsis. Compared with the non-septic group, the 10-year overall survival rate of the septic group was lower (P<0.001). Except for the systemic inflammation response score (SIRS) system, the 10-year overall survival rates of patients in the high risk layers of SOFA (HR=2.50, 95%CI 2.23-2.80, P<0.001), SAPS (HR=2.93, 95%CI 2.64-3.26, P<0.001), SAPS-Ⅱ (HR=2.77, 95%CI 2.51-3.04, P<0.001), APS-Ⅲ (HR=2.90, 95%CI 2.63-3.20, P<0.001), LODS (HR=2.17, 95%CI 1.97-2.38, P<0.001), modified logistic organ dysfunction score (MLODS, HR=2.04, 95%CI 1.86-2.25, P<0.001) and the Oxford acute severity of illness score (OASIS, HR=2.37, 95%CI 2.16-2.60, P<0.001) systems were lower than those in the low risk layers. Conclusion    Compared with SOFA score, APS-Ⅲ score may have higher value in the diagnosis of sepsis in patients who undergo isolated CABG, a valve procedure or a combination of both. Except for SIRS scoring system, SOFA, APS-Ⅲ, SAPS, SAPS-Ⅱ, LODS, MLODS and OASIS scoring systems can be applied to predict the long-term outcome of patients after cardiac surgery.

Objective    To explore the hemodynamic effects of inhaled nitric oxide (iNO) on postoperative hemodynamic in patients with cyanotic congenital heart disease (CHD) combined with decreased pulmonary blood flow. Methods    From 2014 to 2018, there were 1 764 patients who received corrective repair of cyanotic CHD with decreased pulmonary blood flow in the Department of Pediatric Cardiac Surgery of Fuwai Hospital. We included 61 patients with the ratio of right ventricular systolic pressure to systolic blood pressure (SBP) ≥75% after weaning from cardiopulmonary bypass. There were 41 males and 20 females, with the age of 20.5 (9.0, 39.0) months and weight of 12.5±7.8 kg. The patients were divided into two groups: a conventional group (33 patients, conventional therapy only) and a combined therapy group (28 patients, iNO combined with conventional therapy). The hemodynamics during the first 24 hours after iNO therapy and the in-hospital outcomes of the two groups were investigated and compared. Results    There was no statistical difference between the two groups in demographic characteristics and surgical parameters (P>0.05). The hemodynamic effects of iNO within 24 hours included the decrease in the vasoactive inotropic score (VIS, 21.6±6.6 vs. 17.3±7.2, P=0.020) along with the increase in blood pressure (SBP: 73.7±9.7 mm Hg vs. 90.8±9.1 mm Hg, P<0.001) , the decrease in central venous pressure (10.0±3.1 mm Hg vs. 7.9±2.1 mm Hg, P=0.020), the decrease in lactate (2.2±1.7 mmol/L vs. 1.2± 0.5 mmol/L, P<0.001) and increase in urine output [2.8±1.7 mL/(kg·h) vs. 4.9±2.2 mL/(kg·h), P<0.001]. The decrease of VIS at 24 h after the surgery in the conventional therapy group was not statistically significant (22.1±7.9 vs. 20.0±8.5, P=0.232). Besides, we discovered that the need for renal replacement therapy (RRT) was less in the combined therapy group than that in the conventional therapy group, especially in the moderate complicated surgery [risk adjustment in congenital heart surgery (RACHS-1) ≤3] subgroup (9.5% vs. 40.7%, P=0.016). Conclusion    In pediatric patients after corrective repair of cyanotic and pulmonary blood follow decreased CHD with increased pulmonary vascular resistance, iNO combined with conventional therapy can improve the hemodynamics effectively. Compared with the conventional therapy, the combined therapy with iNO can decrease the VIS and the need for RRT, which is beneficial to the postoperative recovery of patients.

Objective    To identify the risk factors of postoperative blood loss among pediatric patients following corrective operation of tetralogy of Fallot (TOF) and to develop nomogram predicting the risk of postoperative blood loss. Methods    A retrospective case-control study was conducted in pediatric TOF patients who underwent corrective operation in our hospital from November 2018 to June 2019. And the clinical data from each enrolled patient were gathered and analyzed. Clinically significant postoperative blood loss was defined as drainage volume from chest tube ≥ 16 mL/kg during the first 24 h after surgery, which corresponded to the 75th percentile of the blood loss in our population. The primary outcome was to determine the independent predictors of postoperative blood loss by the least absolute shrinkage and selection operator (LASSO) regression, univariate and multivariate logistic regression analysis. On the basis of the independent predictors of postoperative bleeding, nomogram was developed and its discrimination and calibration were estimated. Results    A total of 105 children were selected (67 males and 38 females aged 3-72 months). The drainage volume from chest tube in the bleeding group was significantly higher than that in the non-bleeding group during the first 24 h (P<0.000 1). Multivariate logistic regression analysis showed that low body weight (OR=0.538, 95%CI 0.369-0.787, P=0.001), high preoperative hemoglobin concentration (OR=1.036, 95%CI 1.008-1.066, P=0.013) and prolonged intraoperative aortic cross clamp time (OR=1.022, 95%CI 1.000-1.044, P=0.048) were independent risk factors for postoperative blood loss. In the internal validation, the model displayed good discrimination with a C-index of 0.835 (95%CI 0.745-0.926) and high quality of calibration plots in nomogram models was noticed. Conclusion    The nomogram demonstrated good discrimination and calibration in estimating the risk of postoperative blood loss among pediatric patients following corrective operation of TOF.

Objective: To investigate the pathogenic characteristics of viral encephalitis in children living in Hebei province. Methods: We randomly collected cerebrospinal fluid specimens from a total of 399 children diagnosed with viral encephalitis in Hebei Children′s Hospital from May to December 2017. Real-time fluorescence quantitative PCR and Sanger sequencing were used to detect viral nucleic acids in cerebrospinal fluid by an automatic laboratory station. Statistical analysis was performed on the experimental data using SPSS 21.0 software and the clinical data were analyzed. Comparison of infection rates of EV encephalitis in different months, using line × column chi-square test. The MRI and EEG positive rates of different viral encephalitis and viral encephalitis patients not infected with the virus were analyzed by Fisher′s exact probability test. The positive rate of infection with different viruses and non-virus agents was analyzed by Fisher′s exact probability test. Results: The result showed that 80 of 399 samples were positive, and the positive rate was 20.05%. It included 22 cases of enterovirus, 4 cases of influenza A virus, 3 cases of mumps virus, 2 cases of herpes simplex virus type 1, 1 case of herpes simplex virus type 2, 4 cases of EB virus, 7 cases of cytomegalovirus, 7 cases of herpes zoster virus, 8 cases of adenovirus, 14 cases of human herpesvirus type 6. Eight cases had combined viral infection. Eight cases had concurrent infections: 3 cases had enterovirus and herpesvirus type 6 concurrent infection, 1 case had enterovirus and Japanese encephalitis virus concurrent infection and 1 case had herpes simplex virus type 2 and adenovirus, 1 case had influenza A virus herpesvirus type 6, 1 case had mumps virus and herpesvirus type 6, 1 case had mumps virus and herpesvirus type 6, 1 case had herpes simplex virus type 1 and herpes zoster virus concurrent infections. Children with EV viral encephalitis in Hebei Province were highly prevalent in May and June (P=0.016). HHV6 virus encephalitis was more susceptible to infection than non-HHV6 virus (P=0.016); The rate of MRI positive findings in patients with different viral encephalitis was not statistically significant (P>0.05). The result of EEG of different viral encephalitis were P>0.05, which was not statistically significant. Conclusions EV was the most common pathogen of children with viral encephalitis in Hebei province. Encephalitis caused by influenza A virus cannot be ignored in clinical practice.