1.Physicochemical Processes of Biofilm Formation on The Surface of Structures in Water
Kai SHEN ; Fei GAO ; Xu-Qiang HUANG ; Xiao-Peng LU ; Hui-Min ZHOU ; Wei-Rong LI ; Di TIE
Progress in Biochemistry and Biophysics 2024;51(1):145-157
Microorganisms can form biofilms, complex, heterogeneous, multicellular communities that adhere to surfaces. Biofilm formation on the surface of structures in water will accelerate structures’ corrosion, seriously affect their service efficiency and life, and significantly impact the growth of animals, plants, and human life. Hence, clarifying the mechanism of biofilm formation contributes to developing new strategies to control biofilm formation on surface and then reduce infections, biofouling, and contaminations. Biofilm-targeting strategies include the regulation of established biofilms or the modulation of single-cell attachment. In most studies, physicochemical mechanism is frequently applied to explain the initial bacterial adhesion phenomena but rarely to explain other stages of biofilm formation. This review presents a five-step comprehensive description of the physicochemical process from film formation to biofilm maturation: (1) period of film formation; (2) period of bacterial adhesion; (3) period of extracellular-polymeric-substances (EPSs) membrane formation; (4) period of regulating biofilm by quorum sensing (QS); (5) period of biofilm maturation. We first clarify how the film formed by compound molecules affects the surface’s physicochemical properties and initial adhesion, summarizing many factors that affect bacterial adhesion. We then review the types of EPSs and signal molecules secreted by bacteria after irreversible adhesion, as well as their role and QS mechanism in biofilm maturation. Finally, we discuss how bacteria or microcolonies separate from the mature biofilm by physicochemical action and summarize the morphology and adhesion characterization methods after the biofilm matures. This review redefines the role of physicochemical in the whole process of biofilm formation and provides a theoretical basis for the prevention, removal, and utilization of biofilm and other related research fields.
2.Clinical characteristics and prognostic analysis of female patients with Stanford type B aortic dissection.
Tie Nan ZHOU ; Mei Cen LI ; Ya Song WANG ; Hai Wei LIU ; Quan Min JING ; Xiao Zeng WANG ; Ya Ling HAN
Chinese Journal of Cardiology 2023;51(2):172-179
Objective: To explore the clinical characteristics and prognostic factors of female patients with Stanford type B aortic dissection. Methods: This is a single-centre retrospective study. Consecutive patients diagnosed with Stanford type B aortic dissection in General Hospital of Northern Theater Command from June 2002 to August 2021 were enrolled, and grouped based on sex. According to the general clinical conditions and complications of aortic dissection tear, patients were treated with thoracic endovascular aortic repair, surgery, or optimal medication. The clinical characteristics and aortic imaging data of the patients at different stages were collected, adverse events including all-cause deaths, stroke, and occurrence of aortic-related adverse events were obtained during hospitalization and within 30 days and at 1 and 5 years after discharge. According to the time of death, death was classified as in-hospital death, out-of-hospital death, and in-hospital death was divided into preoperative death, intraoperative death and postoperative death. According to the cause of death, death was classified as aortic death, cardiac death and other causes of death. Aortic-related adverse events within 30 days after discharge included new paraplegia, post-luminal repair syndrome, and aortic death; long-term (≥1 year after discharge) aortic-related adverse events included aortic death, recurrent aortic dissection, endoleak and distal ulcer events. The clinical characteristics, short-term and long-term prognosis was compared between the groups. Logistic regression analysis was used to explore the association between different clinical factors and all-cause mortality within 30 days in female and male groups separately. Results: A total of 1 094 patients with Stanford type B aortic dissection were enrolled, mean age was (53.9±12.1) years, and 861 (78.7%) were male and 233 (21.3%) were female. (1) Clinical characteristics: compared with male patients, female patients were featured with older average age, higher proportion of aged≥60 years old, back pain, anemia, optimal medication treatment, and higher cholesterol level; while lower proportion of smoking and drinking history, body mass index, calcium antagonists use, creatine kinase level, and white blood cell count (all P<0.05). However, there was no significant difference in dissection tear and clinical stage, history of coronary heart disease, diabetes, hypertension, and cerebrovascular disease between female and male patients (all P>0.05). (2) Follow-up result: compared with male patients, female patients had a higher rate of 30-day death [6.9% (16/233) vs. 3.8% (33/861), P=0.047], in-hospital death (5.6% (13/233) vs. 2.7% (23/861), P=0.027), preoperative death (3.9% (9/233) vs. 1.5% (12/861), P=0.023) and aorta death (6.0% (14/233) vs. 3.1% (27/861), P=0.041). The 1-year and 5-year follow-up results demonstrated that there were no significant differences in death, cerebrovascular disease, and aorta-related adverse events between the two groups (all P>0.05). (3) Prognostic factors: the results of the univariate logistic regression analysis showed that body mass index>24 kg/m2 (HR=1.087, 95%CI 1.029-1.149, P=0.013), history of anemia (HR=2.987, 95%CI 1.054-8.468, P=0.032), hypertension (HR=1.094, 95%CI 1.047-1.143, P=0.040) and troponin-T>0.05 μg/L (HR=5.818, 95%CI 1.611-21.018, P=0.003)were associated with an increased risk of all-cause mortality within 30 days in female patients. Conclusions: Female patients with Stanford type B aortic dissection have specific clinical characteristics, such as older age at presentation, higher rates of anemia and combined back pain, and higher total cholesterol levels. The risk of death within 1 month is higher in female patients than in male patients, which may be associated with body mass index, hypertension, anemia and troponin-T, but the long-term prognosis for both female and male patients is comparable.
Humans
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Male
;
Female
;
Adult
;
Middle Aged
;
Aged
;
Prognosis
;
Hospital Mortality
;
Retrospective Studies
;
Troponin T
;
Aortic Aneurysm, Thoracic/surgery*
;
Blood Vessel Prosthesis Implantation/adverse effects*
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Treatment Outcome
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Endovascular Procedures/adverse effects*
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Aortic Dissection
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Hypertension/complications*
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Cholesterol
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Risk Factors
4.A monoclonal antibody-based icELISA for puerarin.
Wen-Bin WEI ; Shi-Yan YUE ; Rong-Rong ZHOU ; Tie-Gui NAN ; Min CHEN ; Yuan YUAN
China Journal of Chinese Materia Medica 2022;47(1):48-53
Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.
Animals
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Antibodies, Monoclonal
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Chromatography, Liquid
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Enzyme-Linked Immunosorbent Assay/methods*
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Hybridomas/metabolism*
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Isoflavones
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Mice
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Mice, Inbred BALB C
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Tandem Mass Spectrometry
5.Course of disease and related epidemiological parameters of COVID-19: a prospective study based on contact tracing cohort.
Yan ZHOU ; Wen Jia LIANG ; Zi Hui CHEN ; Tao LIU ; Tie SONG ; Shao Wei CHEN ; Ping WANG ; Jia Ling LI ; Yun Hua LAN ; Ming Ji CHENG ; Jin Xu HUANG ; Ji Wei NIU ; Jian Peng XIAO ; Jian Xiong HU ; Li Feng LIN ; Qiong HUANG ; Ai Ping DENG ; Xiao Hua TAN ; Min KANG ; Gui Min CHEN ; Mo Ran DONG ; Hao Jie ZHONG ; Wen Jun MA
Chinese Journal of Preventive Medicine 2022;56(4):474-478
Objective: To analyze the course of disease and epidemiological parameters of COVID-19 and provide evidence for making prevention and control strategies. Methods: To display the distribution of course of disease of the infectors who had close contacts with COVID-19 cases from January 1 to March 15, 2020 in Guangdong Provincial, the models of Lognormal, Weibull and gamma distribution were applied. A descriptive analysis was conducted on the basic characteristics and epidemiological parameters of course of disease. Results: In total, 515 of 11 580 close contacts were infected, with an attack rate about 4.4%, including 449 confirmed cases and 66 asymptomatic cases. Lognormal distribution was fitting best for latent period, incubation period, pre-symptomatic infection period of confirmed cases and infection period of asymptomatic cases; Gamma distribution was fitting best for infectious period and clinical symptom period of confirmed cases; Weibull distribution was fitting best for latent period of asymptomatic cases. The latent period, incubation period, pre-symptomatic infection period, infectious period and clinical symptoms period of confirmed cases were 4.50 (95%CI:3.86-5.13) days, 5.12 (95%CI:4.63-5.62) days, 0.87 (95%CI:0.67-1.07) days, 11.89 (95%CI:9.81-13.98) days and 22.00 (95%CI:21.24-22.77) days, respectively. The latent period and infectious period of asymptomatic cases were 8.88 (95%CI:6.89-10.86) days and 6.18 (95%CI:1.89-10.47) days, respectively. Conclusion: The estimated course of COVID-19 and related epidemiological parameters are similar to the existing data.
COVID-19
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Cohort Studies
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Contact Tracing
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Humans
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Incidence
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Prospective Studies
6.A multicenter survey of antibiotic use in very and extremely low birth weight infants in Hunan Province.
Ming-Jie WANG ; Shao-Jie YUE ; Jin LIN ; Xi-Rong GAO ; Xiao-Ming PENG ; Meng-Yu CHEN ; Hua-Bao PENG ; Bei CAO ; Yun-Qing ZENG ; Shu-Lian WANG ; Bo WEN ; Xi-Lin HUANG ; Xiao-Ping LI ; Ai-Zhen ZHANG ; Ting CAO ; Yi-Hua CHEN ; Tie-Qiang CHEN ; Chun-Hua YE ; Tao BO ; De-Lin JIANG ; Xiu-Qun HUANG ; Na-Fang REN ; Long-Zhang TAO ; Fang YAO ; Chang-Jun TIAN ; Hong-Ming LI ; Ai-Min ZHANG ; Fu-Rong HUANG ; Wei-Guo ZHANG ; Xiang-Hong CHEN ; Yu-Chan LIU ; Zheng-Lin LIU ; Yan-Shan XU ; Jing-Song MING ; Li CHEN ; Ning-Yi ZHU ; Jun-Min HE ; Sai-Jun YI ; Tuan-Mei WANG ; Zhao-Hui LI ; Gui-Tian WANG
Chinese Journal of Contemporary Pediatrics 2020;22(6):561-566
OBJECTIVE:
To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province.
METHODS:
The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017.
RESULTS:
The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (r=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (r=0.196, P<0.01).
CONCLUSIONS
Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.
Anti-Bacterial Agents
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Birth Weight
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Humans
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Infant
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Infant, Extremely Low Birth Weight
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Infant, Newborn
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Intensive Care Units, Neonatal
;
Surveys and Questionnaires
7.Influence of pre-pregnancy parental body mass index, maternal weight gain during pregnancy, and their interaction on neonatal birth weight.
Xiao-Wei SHI ; Jie YUE ; Min LYU ; Li WANG ; E BAI ; Li-Jun TIE
Chinese Journal of Contemporary Pediatrics 2019;21(8):783-788
OBJECTIVE:
To investigate the influence of pre-pregnancy parental body mass index (BMI), maternal weight gain during pregnancy, and their interaction on neonatal birth weight.
METHODS:
A total of 1 127 pregnant women who underwent regular prenatal examinations and full-term singleton delivery in the First Hospital of Xi'an Jiaotong University from January 2017 to October 2018 were enrolled. The data on their pre-pregnancy BMI, maternal weight gain during pregnancy, pre-pregnancy BMI of the husband, and neonatal birth weight were collected. The interaction between pre-pregnancy parental BMI and maternal weight gain during pregnancy was analyzed, and their correlation with neonatal birth weight was analyzed.
RESULTS:
Among the 1 127 full-term neonates, the detection rates of low birth weight neonates and macrosomia were 2.22% (25/1 127) and 3.82% (43/1 127) respectively. There were significant differences in pre-pregnancy parental BMI and maternal weight gain during pregnancy among the low birth weight, normal birth weight, and macrosomia groups (P<0.05). Neonatal birth weight was positively correlated with pre-pregnancy parental BMI and maternal weight gain during pregnancy (r=0.097-0.322, P<0.05). Low maternal weight before pregnancy increased the risk of low birth weight (RR=4.17, 95%CI: 1.86-9.38), and maternal overweight/obesity before pregnancy (RR=3.59, 95%CI: 1.93-6.67) and excessive weight gain during pregnancy (RR=3.21, 95%CI: 1.39-7.37) increased the risk of macrosomia. No interaction between pre-pregnancy maternal BMI and maternal weight gain during pregnancy was observed.
CONCLUSIONS
Pre-pregnancy parental BMI and maternal weight gain during pregnancy are related to neonatal birth weight, and there is no interaction between pre-pregnancy maternal BMI and maternal weight gain during pregnancy.
Birth Weight
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Body Mass Index
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Female
;
Gestational Weight Gain
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Humans
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Infant, Newborn
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Pregnancy
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Pregnancy Complications
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Risk Factors
;
Weight Gain
8.Efficacy of inactivated autologous porous bone flap and BAM bone-induced artificial bone for repairing skull defect in rats.
Chao-Min WANG ; Tie-Jian LIU ; Zhen-Hua SONG ; Xiao-Yu GUO ; Da-Nian WEI ; Cheng-Yong LIU
Journal of Southern Medical University 2018;38(6):748-754
OBJECTIVETo study the effect of BAM bone grafting combined with inactivated autologous porous bone flap in repairing skull defect in rats.
METHODSSeventy-two Wistar rats with skull defect were randomly divided into control group, inactivated autologous bone flap group (AB group), BAM bone-induced artificial bone material group (BAM group), and inactivated autologous bone flap with BAM bone-induced artificial bone group (BAM+AB group). The bone healing was evaluated with micro-CT and the new bone formation was assessed with histological staining at 1, 2, and 3 months after modeling.
RESULTSInactivated porous bone flap combined with BAM bone-induced artificial bone effectively induced vascular and fibrous tissue regeneration and osteogenesis in the cranial defects. With the inactivated porous bone flap as the scaffold, BAM bone-induced artificial bone obviously promoted the restoration of the skull appearance in the rats with cranial defects.
CONCLUSIONInactivated autologous bone flap group and BAM bone-induced artificial bone material can promote skull healing and restoration of the original skull appearance, and can be used for reconstruction of the local anatomy of the skull surface.
9.Safety and Necessity of Antiplatelet Therapy on Patients Underwent Endovascular Aortic Repair with Both Stanford Type B Aortic Dissection and Coronary Heart Disease.
Rui-Xia HE ; Lei ZHANG ; Tie-Nan ZHOU ; Wen-Jie YUAN ; Yan-Jie LIU ; Wen-Xia FU ; Quan-Min JING ; Hai-Wei LIU ; Xiao-Zeng WANG
Chinese Medical Journal 2017;130(19):2321-2325
BACKGROUNDAcute aortic dissection is known as the most dangerous aortic disease, with management and prognosis determined as the disruption of the medial layer provoked by intramural bleeding. The objective of this study was to evaluate the safety and necessity of antiplatelet therapy on patients with Stanford Type B aortic dissection (TBAD) who underwent endovascular aortic repair (EVAR).
METHODSThe present study retrospectively analyzed 388 patients with TBAD who underwent EVAR and coronary angiography. The primary outcomes were hemorrhage, death, endoleak, recurrent dissection, myocardial infarction, and cerebral infarction in patients with and without aspirin antiplatelet therapy at 1 month and 12 months.
RESULTSOf those 388 patients, 139 (35.8%) patients were treated with aspirin and 249 (64.2%) patients were not treated with aspirin. Patients in the aspirin group were elderly (57.0 ± 10.3 years vs. 52.5 ± 11.9 years, respectively, χ2 = 3.812, P < 0.001) and had more hypertension (92.1% vs. 83.9%, respectively, χ2 = 5.191, P = 0.023) and diabetes (7.2% vs. 2.8%, respectively, χ2 = 4.090, P = 0.043) than in the no-aspirin group. Twelve patients (aspirin group vs. no-aspirin group; 3.6% vs. 2.8%, respectively, χ2 = 0.184, P = 0.668) died at 1-month follow-up, while the number was 18 (4.6% vs. 5.0%, respectively, χ2 = 0.027, P = 0.870) at 12-month follow-up. Hemorrhage occurred in 1 patient (Bleeding Academic Research Consortium [BARC] Type 2) of the aspirin group, and 3 patients (1 BARC Type 2 and 2 BARC Type 5) in the no-aspirin group at 1-month follow-up (χ2 = 0.005, P = 0.944). New hemorrhage occurred in five patients in the no-aspirin group at 12-month follow-up. Three patients in the aspirin group while five patients in the no-aspirin group had recurrent dissection for endoleak at 1-month follow-up (2.3% vs. 2.2%, respectively, χ2 = 0.074, P = 0.816). Four patients had new dissection in the no-aspirin group at 12-month follow-up (2.3% vs. 3.8%, respectively, χ2 = 0.194, P = 0.660). Each group had one patient with myocardial infarction at 1-month follow-up (0.8% vs. 0.4%, respectively, χ2 = 0.102, P = 0.749) and one more patient in the no-aspirin group at 12-month follow-up. No one had cerebral infarction in both groups during the 12-month follow-up. In the percutaneous coronary intervention (PCI) subgroup, 44 (31.7%) patients had taken dual-antiplatelet therapy (DAPT, aspirin + clopidogrel) and the other 95 (68.3%) patients had taken only aspirin. There was no significant difference in hemorrhage (0% vs. 1.1%, respectively, χ2 = 0.144, P = 0.704), death (4.8% vs. 4.5%, respectively, χ2 = 0.154, P = 0.695), myocardial infarction (2.4% vs. 0%, respectively, χ2 = 0.144, P = 0.704), endoleak, and recurrent dissection (0% vs. 3.4%, respectively, χ2 = 0.344, P = 0.558) between the two groups at 12-month follow-up.
CONCLUSIONSThe present study indicated that long-term oral low-dose aspirin was safe for patients with both TBAD and coronary heart disease who underwent EVAR. For the patients who underwent both EVAR and PCI, DAPT also showed no increase in hemorrhage, endoleak, recurrent dissection, death, and myocardial infarction.
10.Safety and Necessity of Antiplatelet Therapy on Patients Underwent Endovascular Aortic Repair with Both Stanford Type B Aortic Dissection and Coronary Heart Disease
He RUI?XIA ; Zhang LEI ; Zhou TIE?NAN ; Yuan WEN?JIE ; Liu YAN?JIE ; Fu WEN?XIA ; Jing QUAN?MIN ; Liu HAI?WEI ; Wang XIAO?ZENG
Chinese Medical Journal 2017;(19):2321-2325
Background: Acute aortic dissection is known as the most dangerous aortic disease, with management and prognosis determined as the disruption of the medial layer provoked by intramural bleeding. The objective of this study was to evaluate the safety and necessity of antiplatelet therapy on patients with Stanford Type B aortic dissection (TBAD) who underwent endovascular aortic repair (EVAR). Methods: The present study retrospectively analyzed 388 patients with TBAD who underwent EVAR and coronary angiography. The primary outcomes were hemorrhage, death, endoleak, recurrent dissection, myocardial infarction, and cerebral infarction in patients with and without aspirin antiplatelet therapy at 1 month and 12 months. Results: Of those 388 patients, 139 (35.8%) patients were treated with aspirin and 249 (64.2%) patients were not treated with aspirin. Patients in the aspirin group were elderly (57.0 ± 10.3 years vs. 52.5 ± 11.9 years, respectively, χ2 = 3.812, P < 0.001) and had more hypertension (92.1% vs. 83.9%, respectively, χ2 = 5.191, P = 0.023) and diabetes (7.2% vs. 2.8%, respectively, χ2 = 4.090, P = 0.043) than in the no?aspirin group. Twelve patients (aspirin group vs. no?aspirin group; 3.6% vs. 2.8%, respectively, χ2 = 0.184, P = 0.668) died at 1?month follow?up, while the number was 18 (4.6% vs. 5.0%, respectively, χ2 = 0.027, P = 0.870) at 12?month follow?up. Hemorrhage occurred in 1 patient (Bleeding Academic Research Consortium [BARC] Type 2) of the aspirin group, and 3 patients (1 BARC Type 2 and 2 BARC Type 5) in the no?aspirin group at 1?month follow?up ( χ2 = 0.005, P = 0.944). New hemorrhage occurred in five patients in the no?aspirin group at 12?month follow?up. Three patients in the aspirin group while five patients in the no?aspirin group had recurrent dissection for endoleak at 1?month follow?up (2.3% vs. 2.2%, respectively, χ2 = 0.074, P = 0.816). Four patients had new dissection in the no?aspirin group at 12?month follow?up (2.3% vs. 3.8%, respectively, χ2 = 0.194, P = 0.660). Each group had one patient with myocardial infarction at 1?month follow?up (0.8% vs. 0.4%, respectively, χ2 = 0.102, P = 0.749) and one more patient in the no?aspirin group at 12?month follow?up. No one had cerebral infarction in both groups during the 12?month follow?up. In the percutaneous coronary intervention (PCI) subgroup, 44 (31.7%) patients had taken dual?antiplatelet therapy (DAPT, aspirin + clopidogrel) and the other 95 (68.3%) patients had taken only aspirin. There was no significant difference in hemorrhage (0% vs. 1.1%, respectively,χ2 = 0.144, P = 0.704), death (4.8% vs. 4.5%, respectively, χ2 = 0.154, P = 0.695), myocardial infarction (2.4% vs. 0%, respectively,χ2 = 0.144, P = 0.704), endoleak, and recurrent dissection (0% vs. 3.4%, respectively, χ2 = 0.344, P = 0.558) between the two groups at 12?month follow?up. Conclusions: The present study indicated that long?term oral low?dose aspirin was safe for patients with both TBAD and coronary heart disease who underwent EVAR. For the patients who underwent both EVAR and PCI, DAPT also showed no increase in hemorrhage, endoleak, recurrent dissection, death, and myocardial infarction.

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