Objective:To explore the predictive value of preoperative CT angiography (CTA) for incomplete endothelialization of the occluder after left atrial appendage closure (LAAC) in patients with atrial fibrillation.Methods:The clinical data of 92 atrial fibrillation patients underwent LAAC in the Tangdu Hospital of Air Force Military Medical University from January 2022 to June 2024 were retrospectively analyzed. CTA examinations were performed both before operation and 3 months after operation. Before operation, the long diameter of left atrial appendage opening, short diameter of left atrial appendage opening, area of left atrial appendage opening, diameter of anchoring area and effective depth were measured. After operation, the condition of occluder endothelialization was evaluated, and the patients were divided into the completely endothelialization group and the incomplete endothelialization group. Multivariate Logistic regression analysis was used to analyze the independent risk factors of occluder incomplete endothelialization after LAAC in patients with atrial fibrillation. The predictive value of independent risk factors was evaluated by the receiver operating characteristic (ROC) curve.Results:Among the 92 patients, CTA 3 months after the operation showed that 58 cases had complete occluder endothelialization (complete endothelialization group), and 34 cases had occluder incomplete endothelialization (incomplete endothelialization group). Before operation, the long diameter of left atrial appendage opening, short diameter of left atrial appendage opening, area of left atrial appendage opening and diameter of anchoring area in incomplete endothelialization group were significantly larger than those in complete endothelialization group: (28.35 ± 1.77) mm vs. (26.21 ± 2.21) mm, (22.09 ± 2.01) mm vs. (20.86 ± 1.75) mm, (512.76 ± 63.35) mm 2 vs. (453.83 ± 75.39) mm 2 and (24.71 ± 2.50) mm vs. (23.12 ± 2.40) mm, and there were statistical differences ( P<0.01); there was no statistical difference in effective depth between the two groups ( P>0.05). Multivariate Logistic regression analysis result showed that the long diameter of left atrial appendage opening before operation was an independent risk factor for occluder incomplete endothelialization after LAAC in patients with atrial fibrillation ( OR = 2.141, 95% CI 1.217 to 3.768, P<0.01). ROC curve analysis result showed that the area under the curve of the long diameter of left atrial appendage opening before operation for predicting occluder incomplete endothelialization after LAAC in patients with atrial fibrillation was 0.768 (95% CI 0.674 to 0.862, P<0.01), the optimal cut-off value was 26.5 mm, the sensitivity was 88.2%, and the specificity was 55.2%. Conclusions:A larger long diameter of left atrial appendage opening before operation can lead to occluder incomplete endothelialization after LAAC in patients with atrial fibrillation.

Objective:To establish a preliminary performance validation protocol for the bioinformatics procedure of metagenomic next-generation sequencing (mNGS) in clinical laboratories.Methods:Three types of simulated datasets were designed and the CatⅠ dataset mainly consisted of pathogen reference genomes and human sequences. CatⅠA was a dataset composed of common pathogens mixed with human sequences and was used to evaluate the inclusiveness, accuracy, recall rates, precision, F1-Score, and other indicators of the mNGS bioinformatics procedure. CatⅠB was a dataset composed of closely related species of common pathogens mixed with human sequences, which was used to evaluate the discriminating ability of closely related species of bioinformatics procedure by calculating the detection rates and the relative abundance ratio of closely related species. The real data of 200 clinical samples was selected to construct CatⅡ and the simulated dataset consisted of colonized bacteria, experimental environment bacteria, reagent engineering bacteria, pathogen reference genomes, and human sequences, which was used to evaluate the sensitivity, specificity, and accuracy of bioinformatics pipeline for pathogens detection. The CatⅢ dataset was obtained from the negative bronchoalveolar lavage fluid BALF sequencing data mixed with 20 rare pathogens sequences in order to evaluate the positive detection rates and recall rates of rare pathogens in the bioinformatics analysis.Results:The analysis of the CatⅠA dataset showed that the positive consistency rate, inclusiveness, precision and accuracy of the bioinformatics peocedure under three sequence gradients were all greater than 99%, with a recall rate of 72.31% (95% CI 69.61%-75.01%) and a F1 Score of 82.00% (95% CI 79.77%-84.22%). In the CatⅠB dataset, the closely related species could be effectively detected at all sequence and proportion gradients, and the relative abundance ratio of closely related species was within 2 times of the design ratio except for the coronavirus, haemophilus, primate bocaparvovirus, human respiratory syncytial virus, and eimeria, indicating good ability to identify the closely related species. All the 24 species of pathogens included in CatⅡ dataset were effectively detected, with the sensitivity, specificity, and accuracy all greater than 90%. All rare pathogens were detected in the CatⅢ dataset, with a detection rate of 100%. Conclusions:With the simulated datasets, the performance validation scheme for the mNGS bioinformatics analysis was preliminary established and could evaluate the accuracy of sequence classification, the ability to identify the closely related species, and detection ability of common and rare pathogens, which may provide some references for the construction of mNGS process.

Objective To establish a performance verification scheme for the metagenomic next-generation sequencing(mNGS)DNA workflow.Methods Reference materials and clinical samples were used for conducting experiments.The mNGS detection results were evaluated in terms of limit of detection(LOD),repeatability,robustness,anti-interference ability,specificity and accuracy,as well as the patterns of library construction and the performance of sequencers.Results All species in the reference materials were stably detected,and the LOD of mNGS was 5.0E+02 CFU/mL(copies/mL).The repeatability was 100％and the within-batch(coefficient of variation)CV ranged from 8.53％to 38.73％.The linear correlation coefficient|r|＞0.9 was found between the input pathogenic microorganism concentration and the read count.Meanwhile,the experimental robustness was found to be good.The results of the anti-interference test showed that the higher concentration of human DNA inputted,the fewer pathogenic microorganism read counts detected by mNGS.Meanwhile,the read counts of related species presented a proportional relationship with the corresponding pathogenic microorganisms concentration inputted,which meant the validation of the cross-interference test had been passed.Furthermore,the detection result of D0 was negative.The accuracy of clinical samples testing was 90.9％(10/11).In addition,the library quality control results obtained by the automatic liquid handling workstation and manually operation were all acceptable.The performance of the three Illumina sequencers met or were better than the factory standards.Conclusion The clinical laboratory performance verification scheme for mNGS detection was established,which included the design for reference materials,comparison of different patterns for library construction,and performance evaluation of the sequencers.More importantly,the performance verification scheme can be used to evaluate and ensure the quality of mNGS DNA workflow detection process.

Objective To establish a performance verification scheme for the metagenomic next-generation sequencing(mNGS)DNA workflow.Methods Reference materials and clinical samples were used for conducting experiments.The mNGS detection results were evaluated in terms of limit of detection(LOD),repeatability,robustness,anti-interference ability,specificity and accuracy,as well as the patterns of library construction and the performance of sequencers.Results All species in the reference materials were stably detected,and the LOD of mNGS was 5.0E+02 CFU/mL(copies/mL).The repeatability was 100％and the within-batch(coefficient of variation)CV ranged from 8.53％to 38.73％.The linear correlation coefficient|r|＞0.9 was found between the input pathogenic microorganism concentration and the read count.Meanwhile,the experimental robustness was found to be good.The results of the anti-interference test showed that the higher concentration of human DNA inputted,the fewer pathogenic microorganism read counts detected by mNGS.Meanwhile,the read counts of related species presented a proportional relationship with the corresponding pathogenic microorganisms concentration inputted,which meant the validation of the cross-interference test had been passed.Furthermore,the detection result of D0 was negative.The accuracy of clinical samples testing was 90.9％(10/11).In addition,the library quality control results obtained by the automatic liquid handling workstation and manually operation were all acceptable.The performance of the three Illumina sequencers met or were better than the factory standards.Conclusion The clinical laboratory performance verification scheme for mNGS detection was established,which included the design for reference materials,comparison of different patterns for library construction,and performance evaluation of the sequencers.More importantly,the performance verification scheme can be used to evaluate and ensure the quality of mNGS DNA workflow detection process.

Objective:To explore the predictive value of preoperative CT angiography (CTA) for incomplete endothelialization of the occluder after left atrial appendage closure (LAAC) in patients with atrial fibrillation.Methods:The clinical data of 92 atrial fibrillation patients underwent LAAC in the Tangdu Hospital of Air Force Military Medical University from January 2022 to June 2024 were retrospectively analyzed. CTA examinations were performed both before operation and 3 months after operation. Before operation, the long diameter of left atrial appendage opening, short diameter of left atrial appendage opening, area of left atrial appendage opening, diameter of anchoring area and effective depth were measured. After operation, the condition of occluder endothelialization was evaluated, and the patients were divided into the completely endothelialization group and the incomplete endothelialization group. Multivariate Logistic regression analysis was used to analyze the independent risk factors of occluder incomplete endothelialization after LAAC in patients with atrial fibrillation. The predictive value of independent risk factors was evaluated by the receiver operating characteristic (ROC) curve.Results:Among the 92 patients, CTA 3 months after the operation showed that 58 cases had complete occluder endothelialization (complete endothelialization group), and 34 cases had occluder incomplete endothelialization (incomplete endothelialization group). Before operation, the long diameter of left atrial appendage opening, short diameter of left atrial appendage opening, area of left atrial appendage opening and diameter of anchoring area in incomplete endothelialization group were significantly larger than those in complete endothelialization group: (28.35 ± 1.77) mm vs. (26.21 ± 2.21) mm, (22.09 ± 2.01) mm vs. (20.86 ± 1.75) mm, (512.76 ± 63.35) mm 2 vs. (453.83 ± 75.39) mm 2 and (24.71 ± 2.50) mm vs. (23.12 ± 2.40) mm, and there were statistical differences ( P<0.01); there was no statistical difference in effective depth between the two groups ( P>0.05). Multivariate Logistic regression analysis result showed that the long diameter of left atrial appendage opening before operation was an independent risk factor for occluder incomplete endothelialization after LAAC in patients with atrial fibrillation ( OR = 2.141, 95% CI 1.217 to 3.768, P<0.01). ROC curve analysis result showed that the area under the curve of the long diameter of left atrial appendage opening before operation for predicting occluder incomplete endothelialization after LAAC in patients with atrial fibrillation was 0.768 (95% CI 0.674 to 0.862, P<0.01), the optimal cut-off value was 26.5 mm, the sensitivity was 88.2%, and the specificity was 55.2%. Conclusions:A larger long diameter of left atrial appendage opening before operation can lead to occluder incomplete endothelialization after LAAC in patients with atrial fibrillation.

Objective:To establish a preliminary performance validation protocol for the bioinformatics procedure of metagenomic next-generation sequencing (mNGS) in clinical laboratories.Methods:Three types of simulated datasets were designed and the CatⅠ dataset mainly consisted of pathogen reference genomes and human sequences. CatⅠA was a dataset composed of common pathogens mixed with human sequences and was used to evaluate the inclusiveness, accuracy, recall rates, precision, F1-Score, and other indicators of the mNGS bioinformatics procedure. CatⅠB was a dataset composed of closely related species of common pathogens mixed with human sequences, which was used to evaluate the discriminating ability of closely related species of bioinformatics procedure by calculating the detection rates and the relative abundance ratio of closely related species. The real data of 200 clinical samples was selected to construct CatⅡ and the simulated dataset consisted of colonized bacteria, experimental environment bacteria, reagent engineering bacteria, pathogen reference genomes, and human sequences, which was used to evaluate the sensitivity, specificity, and accuracy of bioinformatics pipeline for pathogens detection. The CatⅢ dataset was obtained from the negative bronchoalveolar lavage fluid BALF sequencing data mixed with 20 rare pathogens sequences in order to evaluate the positive detection rates and recall rates of rare pathogens in the bioinformatics analysis.Results:The analysis of the CatⅠA dataset showed that the positive consistency rate, inclusiveness, precision and accuracy of the bioinformatics peocedure under three sequence gradients were all greater than 99%, with a recall rate of 72.31% (95% CI 69.61%-75.01%) and a F1 Score of 82.00% (95% CI 79.77%-84.22%). In the CatⅠB dataset, the closely related species could be effectively detected at all sequence and proportion gradients, and the relative abundance ratio of closely related species was within 2 times of the design ratio except for the coronavirus, haemophilus, primate bocaparvovirus, human respiratory syncytial virus, and eimeria, indicating good ability to identify the closely related species. All the 24 species of pathogens included in CatⅡ dataset were effectively detected, with the sensitivity, specificity, and accuracy all greater than 90%. All rare pathogens were detected in the CatⅢ dataset, with a detection rate of 100%. Conclusions:With the simulated datasets, the performance validation scheme for the mNGS bioinformatics analysis was preliminary established and could evaluate the accuracy of sequence classification, the ability to identify the closely related species, and detection ability of common and rare pathogens, which may provide some references for the construction of mNGS process.

Objective:To investigate the effect of astragaloside IV (AS-IV) regulating the signal axis of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) on the mobilization of bone marrow endothelial progenitor cells (EPCs) to peripheral blood in diabetes skin ulcer (DSU) rats.Methods:Twenty four SPF grade male Sprague Dawley (SD) rats were selected to make the model of type 2 diabetes rats by intraperitoneal injection of 30 mg/kg 1% (plastid ratio) streptozotocin, and then round full-thickness skin with a diameter of 2 cm was cut on both sides of the waist and back to make the skin ulcer model of diabetes rats. After that, they were randomly divided into AS-IV group (50 mg/kg AS-IV), blocker group (50 mg/kg AS-IV+ 5 mg/kg AMD3100) and model group. At the same time, a blank group ( n=8) was set up, The drug was administered via intraperitoneal injection, and the model group and blank group were treated with 0.9% NaCl of equal volume. On the 10th day, peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats were collected. The number of EPCs in peripheral blood of each group of rats was measured by flow cytometry, and the protein expression of SDF-1α and CXCR4 in peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats was detected by enzyme-linked immunosorbent assay (ELISA); At the same time, the wound healing rates of each group were tested. Results:On the 10th and 21st day after modeling, the wound healing rate of each group of rats was compared. The blank group healed the fastest, while the model group healed the slowest. The AS-IV group had better healing than the model group and the blocker group, and the difference was statistically significant (all P<0.05). On the 10th day after modeling, the positive rates of peripheral blood EPCs in the white group, AS-IV group, and blocker group were significantly higher than those in the model group (all P<0.05), while the positive rates of peripheral blood EPCs in the blocker group were significantly lower than those in the AS-IV group (all P<0.05). On the 10th day after modeling, the protein expression of SDF-1α and CXCR4 in the wound, serum, and bone marrow of the model group was the lowest, while the protein expression in the blank group was the highest (all P<0.05). The protein expression of SDF-1α and CXCR4 in the wound, serum, bone marrow of the AS-IV group was significantly higher than that of the blocker group and model group, and the difference was statistically significant (all P<0.05). Conclusions:Astragaloside IV can promote the mobilization and migration of endothelial progenitor cells from bone marrow to peripheral blood in diabetes ulcer rats by regulating SDF-1α/CXCR4 signal axis, and can participate in angiogenesis of diabetes ulcer wounds as seed cells to promote the healing of diabetes skin ulcers.

Objective:To investigate the diagnostic value of metagenomic next-generation sequencing (mNGS) in AIDS patients complicated with Pneumocystis jirovecii ( P. jirovecii) infection. Methods:This is a retrospective study. From January 2019 to June 2021, the respiratory tract and other body fluid samples of 236 cases of AIDS co-infected patients diagnosed in the AIDS Department of Changsha First Hospital were collected, along with corresponding medical histories. Traditional etiological hexamine silver staining and serum 1,3-β-D glucan (BDG) were performed simultaneously with mNGS detection, and Fisher′s exact test was used to analyze the results and compare the diagnostic performances of mNGS with those of hexamine silver staining and serum G test.Results:A total of 236 cases of AIDS patients with pulmonary infection were collected and tested. Seventy-seven cases were clinically diagnosed with Pneumocystis jiroveci pneumonia and 159 cases with non- Pneumocystis jiroveci pneumonia. Among the 236 AIDS patients with pulmonary infection, mNGS detected 77 [32.63%(77/236)] positive cases of Pneumocystis jiroveci, while hexamine silver staining detected 10[4.24%(10/236)] and serum BDG detected 146 [61.86% (146/236). Based on these clinical diagnostic results, the sensitivity of mNGS detection was 100% (77/77) for the 77 patients with Pneumocystis pneumoniae, significantly higher than that of silver hexamine staining [12.99% (10/77), P=0.046] and serum BDG [58.44% (45/77), P=0.038]. The mNGS showed good specificity, which was the same as that of hexamine silver staining [100% (159/159)] and significantly higher than that of serum BDG [36.48% (58/159), P=0.026]. With therapeutic clinical diagnosis as the reference method, the accuracy of mNGS detection was 100% (236/236). Conclusions:This study evaluated the diagnostic value of mNGS detection in AIDS patients with Pneumocystis jirovecii infection. The results showed that the sensitivity and specificity of mNGS detection were high, and it had exceptional clinical application value in the pathogenic detection of infectious diseases.

Objective:To evaluate the efficacy of allogeneic hematopoietic cell transplantation(allo-HSCT) using unrelated cord blood or haploidentical donors in the treatment of children with primary immunodeficiency diseases (PID).Methods:The clinical data of 60 children with PID admitted to Chinese People′s Liberation Army General Hospital-Sixth Medical Center from April 2014 to October 2019 were retrospectively analyzed, including 56 cases of chronic granulomatous disease, 2 cases of severe combined immunodeficiency disease, 1 case of high-IgM syndrome and 1 case of severe congenital neutropenia.All patients underwent allo-HSCT, including 12 cases receiving the transplantation from unrelated cord blood (UCB group) and 48 cases from haploidentical donors combined with a third party unrelated cord blood (haploid group). Among these patients, there were 59 males and 1 female, with a median age of 3.4 years.All patients received a myeloablative conditioning regimen based on Busulfan.The prophylaxis of acute graft versus host disease (aGVHD) was performed based on Cyclosporine.In the UCB group, the median dose of mononuclear cells and CD 34+ cells was 0.67×10 8/kg and 0.51×10 6/kg recipient body weight, respectively; In the haploid group, bone marrow and peripheral stem cells from haploid donors were infused on day 01 and day 02, respectively.The third party cord blood was infused 4 hours before bone marrow infusion.The median dose of mononuclear cells and CD 34+ cells of bone marrow and peripheral stem cells from haploid donors was 9.97×10 8/kg and 5.12×10 6/kg recipient body weight, respectively.Kaplan-Meier method was used to analyze the overall survival rate. Results:The median day to neutrophil and platelet engraftment was 13.0 days and 23.5 days, respectively.The rate of complete donor chime-rism was shown 30.0 days after transplantation.There was no case with primary engraftment failure, and 1 case with secondary engraftment failure.The incidence of grade Ⅰ-Ⅱ and grade Ⅲ-Ⅳ aGVHD was 43.3% and 15.5%, respectively.The incidence of chronic graft versus host disease with limited skin type was 6.7%, while that with extensive type was 1.1%.The median follow-up period was 818 days.There were 6 death cases, among which, 5 cases died from infection and 1 case died from heart failure.The total mortality related to transplantation was 11.9%.A total of 53 cases survived without diseases.The estimated 5-year failure free survival and overall survival rate was 83.9% and 88.1%, respectively.Conclusion:The efficacy of allo-HSCT in the treatment of children with PID using unrelated cord blood and haploidentical donors is favorable.

Objective:Acute kidney injury (AKI) is one of the common complications in critically ill septic patients, which is associated with increased risks of death, cardiovascular events, and chronic renal dysfunction. The duration of AKI and the renal function recovery status after AKI onset can affect the patient prognosis. Nevertheless, it remains controversial whether early recovery status after AKI is closely related to the prognosis in patients with sepsis-associated AKI (SA-AKI). In addition, early prediction of renal function recovery after AKI is beneficial to individualized treatment decision-making and prevention of severe complications, thus improving the prognosis. At present, there is limited clinical information on how to identify SA-AKI patients at high risk of unrecovered renal function at an early stage. The study aims to investigate the association between early recovery status after SA-AKI, identify risk factors for unrecovered renal function, and to improve patients ' quality of life.Methods:We retrospectively analyzed clinical data of septic patients who were admitted to the intensive care unit (ICU) and developed AKI within the first 48 hours after ICU admission in the Second Xiangya Hospital and the Third Xiangya Hospital of Central South University from January 2015 to March 2017. Sepsis was defined based on the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). AKI was diagnosed and staged according to the 2012 Kidney Disease:Improving Global Outcomes (KDIGO) guideline. SA-AKI patients were assigned into 3 groups including a complete recovery group, a partial recovery group, and an unrecovered group based on recovery status at Day 7 after the diagnosis of AKI. Patients ' baseline characteristics were collected, including demographics, comorbidities, clinical and laboratory examination information at ICU admission, and treatment within the first 24 hours. The primary outcome of the study was the composite of death and chronic dialysis at 90 days, and secondary outcomes included length of stay in the ICU, length of stay in the hospital, and persistent renal dysfunction. Multivariate regression analysis was performed to evaluate the prognostic value of early recovery status after AKI and to determine the risk factors for unrecovered renal function after AKI. Sensitivity analysis was conducted in patients who still stayed in hospital on Day 7 after AKI diagnosis, patients without premorbid chronic kidney disease, and patients with AKI Stage 2 to 3.Results:A total of 553 SA-AKI patients were enrolled, of whom 251 (45.4％), 73 (13.2％), and 229 (41.4％) were categorized as the complete recovery group, the partial recovery group, and the unrecovered group, respectively. Compared with the complete or partial recovery group, the unrecovered group had a higher incidence of 90-day mortality (unrecovered vs partial recovery or complete recovery: 64.2％ vs 26.0％ or 22.7％; P<0.001) and 90-day composite outcome (unrecovered vs partial recovery or complete recovery:65.1％vs 27.4％or 22.7％;P<0.001). The unrecovered group also had a shorter length of stay in the hospital and a larger proportion of progression into persistent renal dysfunction than the other 2 groups. After adjustment for potential confounders, patients in the unrecovered group were at an increased risk of 90-day mortality (HR=3.50, 95％ CI 2.47 to 4.96, P<0.001) and 90-day composite outcome (OR=5.55, 95％CI 3.43 to 8.98, P<0.001) when compared with patients in the complete recovery group, but patients in the partial recovery group had no significant difference (P>0.05). Male sex, congestive heart failure, pneumonia, respiratory rate>20 beats per minute, anemia, hyperbilirubinemia, need for mechanical ventilation, and AKI Stage 3 were identified as independent risk factors for unrecovered renal function after AKI. The sensitivity analysis further supported that unrecovered renal function after AKI remained an independent predictor for 90-day mortality and composite outcome in the subgroups. Conclusion:The early recovery status after AKI is closely associated with poor prognosis in critically ill patients with SA-AKI. Unrecovered renal function within the first 7 days after AKI diagnosis is an independent predictor for 90-day mortality and composite outcome. Male sex, congestive heart failure, pneumonia, tachypnea, anemia, hyperbilirubinemia, respiratory failure, and severe AKI are risk factors for unrecovered renal function after AKI. Therefore, timely assessment for the renal function in the early phase after AKI diagnosis is essential for SA-AKI patients. Furthermore, patients with unrecovered renal function after AKI need additional management in the hospital, including rigorous monitoring, avoidance of nephrotoxin, and continuous assessment for the renal function, and after discharge, including more frequent follow-up, regular outpatient consultation, and prevention of long-term adverse events.