1.Based on 16S rDNA Technology and TLRs/MyD88/NF-κB Signaling Pathway, Molecular Mechanism of Shenling Baizhusan Resistance to Diarrhea Irritable Bowel Syndrome Rats Was Investigated
Tengfei LYU ; Jingyu WANG ; Mingyue XIE ; Bin XI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(11):13-22
ObjectiveBased on 16S rDNA technology and molecular biology methods, the molecular mechanism of Shenling Baizhusan in the treatment of diarrhea-predominant irritable bowel syndrome (IBS-D) was investigated. MethodsThe 42 SD rats with SPF were randomly divided into no control group, SLBZS-H, medium (SLBZS-M), low (SLBZS-L) dose group, positive control group and model group, with 7 rats in each group. The rat model of IBS-D was prepared by ice-cold senna (0.45 g∙mL-1) gavage (10 mL∙kg-1) combined with restraint stress for 14 consecutive days. After successful modeling, the corresponding drugs were given to each group with a gavage volume of 10 mL∙kg-1: The positive group was administered with 2.36 , 1.18, 0.59 g∙mL-1 of Shenling Baizhusan in the Positive group and the Model group with the same volume of normal saline for 14 d. The general condition of the rats: Weight, feces, mental state and death were observed and recorded. The body weight, abdominal wall retraction reflex score (AWR) and loose stool rate of rats in each group were measured before (the first day), after the model (day 14) and after treatment (day 28). Hematoxylin-eosin staining was used to observe the morphological characteristics of colon tissues of experimental animals. Enzyme-linked immunosorbent assay was used to quantitatively analyze the concentration of inflammatory mediators in the peripheral blood of experimental animals. Western blotting was used to detect the expression levels of key proteins of Toll-like receptor 4 (TLR4), Toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88) and nuclear factor-κB (NF-κB) signaling pathway in rat colon tissue. 16S rDNA technology was used to detect the structural changes of intestinal microbiota in rats. ResultsCompared with Control, the colon of the Model group showed partial mucosal epithelial shedding and inflammatory cell infiltration. The contents of TNF-α, IL-1β, IL-6 and 5-HT in serum increased (P<0.05), the protein expressions of TLR2, TLR4, MyD88 and NF-κB in colon tissue increased (P<0.05), the diversity indices of Richness, Chao1, abundance-based coverage estimator(ACE) and Shannon decreased (P<0.05), and the phylum Firmicutes, Actinobacteria, The relative richness of Bacteroides-H, Lactobacillus and Ligilactobacillus decreased (P<0.05), while the relative richness of Bacteroidetes, Proteobacteria and Prevotella increased (P<0.05). Compared with the model group, the colonic structure and organization of the SLBZS-H group, SLBZS-M group, SLBZS-L group and Positive group were clearer, and only a small number of inflammatory cells were present in some areas, and the serum contents of TNF-α, IL-1β, IL-6 and 5-HT were decreased (P<0.05), TLR2, TLR4, The protein expressions of MyD88 and NF-κB decreased (P<0.05), and compared with the model group, the diversity indices of Richness, Chao1, ACE and Shannon in the SLBZS-H, SLBZS-M and SLBZS-L groups increased (P<0.05), and the richness of Firmicutes and Actinobacteria increased (P<0.05). The richness of Proteobacteria and Prevotella decreased (P<0.05), and the abundance of Prevotella decreased (P<0.05), Bacteroides-H, Muribaculum, Lactobacillus and salivarius in the Positive group salivarius (P<0.05). ConclusionShenling Baizhusan can effectively treat IBS-D, and its molecular mechanism may be to play a therapeutic role by improving intestinal flora and inhibiting the TLRS/MyD88/NF-κB signaling pathway to reduce inflammatory response.
2.Intelligent blood logistics reinvention: HFMEA-applied transport pathway optimization for biopharmaceutical safety assurance
Qiming YING ; Fangfang JIN ; Fengmin XU ; Jiaji HU ; Danni SONG ; Bin WU ; Qinhong XU ; Dingfeng LYU
Chinese Journal of Blood Transfusion 2026;39(1):123-127
Objective: To explore the application effectiveness of healthcare failure mode and effect analysis (HFMEA) in optimizing intelligent blood logistics transport pathways for safety assurance. Methods: Data from 1 851 cases of intelligent blood logistics transport were collected between September 2023 and March 2025. Based on the implementation phases of HFMEA measures, the cases were divided into a control group (n=120), observation group 1 (n=219), and observation group 2 (n=1 512). Through systematic analysis of the transport processes, hazard scoring and decision tree analysis were conducted for each process, and phased optimization measures were implemented for high-risk failure modes. Results: The transport duration of intelligent blood logistics was 35.5 (20.8, 71.1) min in the control group, 25.1 (10.9, 40.7) min in observation group 1, and 9.9 (4.2, 44.5) min in observation group 2. Observation group 2 exhibited significantly shorter transport time compared to both observation group 1 and the control group, with statistically significant differences between groups (P<0.000 1). Conclusion: The implementation of HFMEA-driven measures significantly reduced intelligent blood logistics transport duration, thereby fostering the evolution of smart hospital ecosystems while enhancing healthcare service quality and operational efficiency.
3.Effect of measurement site on diagnostic performance of CT-derived fractional flow reserve
Yutao ZHOU ; Na ZHAO ; Yunqiang AN ; Lei SONG ; Chaowei MU ; Jingang CUI ; Tao JIANG ; Li XU ; Hongjie HU ; Lin LI ; Dumin LI ; Wenqiang CHEN ; Lijuan FAN ; Feng ZHANG ; Yang GAO ; Bin LYU
Chinese Journal of Radiology 2025;59(6):704-711
Objective:To investigate the effect of CT-derived fractional flow reserve (CT-FFR) measurement sites on the values and the diagnostic performance, and to determine the optimal measurement site for CT-FFR using invasive FFR as the reference standard.Methods:This study was part of the CT-FFR CHINA clinical trial. Patients with suspected coronary artery disease who were scheduled for invasive coronary angiography (ICA) were prospectively recruited from five clinical centers across the country from November 2018 to March 2020. Each enrolled patient underwent coronary CT angiography (CCTA), CT-FFR, ICA, and invasive pressure wire-based FFR assessments sequentially within one week. Four groups of CT-FFR values were obtained on each enrolled target vessels according to different CT-FFR measurement locations: 1, 2, 3 cm distal to the target lesion, and terminal vessel groups. Spearman and Bland-Altman analyses were used to explore the correlation and consistency of CT-FFR values and FFR values at different measurement sites. The measurement deviation of CT-FFR was also compared. Diagnostic accuracy and performance of CT-FFR, including sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve (AUC), in discriminating myocardial ischemia were analyzed across all measurement site groups on a per-vessel level, using FFR as the reference standard.Results:A total of 289 patients with 345 target lesion vessels were included. According to CCTA, there were 51 target vessels (14.8%) with<50% stenosis, 106 vessels (30.7%) with 50%-69% stenosis, and 188 vessels (54.5%) with stenosis≥70%. At per-vessel level, CT-FFR and FFR values at each measurement position group were highly positively correlated: 1 cm distal to target lesion group, r=0.734 ( P<0.001); 2 cm distal to target lesion group, r=0.732 ( P<0.001); 3 cm distal to target lesion group, r=0.737 ( P<0.001); terminal vessel group was 0.719 ( P<0.001). At per-vessel level, CT-FFR and FFR values of all measurement sites were in good agreement (Bland-Altman analysis results): 1 cm distal to target lesion group, 0.014 (95% LoA 0.002-0.026); 2 cm distal to target lesion group, 0.026 (95% LoA 0.015-0.038); 3 cm distal to target lesion group, 0.040 (95% LoA 0.039-0.051); terminal vessel group, 0.075 (95% LoA 0.064-0.087). And at per-vessel level, the accuracy of diagnosing myocardial ischemia with CT-FFR at 1 cm was highest [84.6% (95% CI 80.4%-88.3%)], and the lowest accuracy in the terminal vessel group [67.0% (95% CI 61.7%-72.0%)]. However, there was no significant difference in the diagnostic accuracy of CT-FFR at 1 cm, 2 cm [80.6% (95% CI 76.1%-84.6%)] and 3 cm [77.5% (95% CI 72.6%-81.7%)]. AUC of CT-FFR at 1 cm distal to the lesion were both highest for global level and moderately stenosis (50%-69%) lesions [0.85 (95% CI 0.81-0.89), 0.84 (95% CI 0.77-0.90)]. And the differences were statistically significant among the four measurement location groups (all P<0.05). Conclusions:The deviation of CT-FFR increases with measurement site distance distal to target lesions. One centimeter distal to the target lesion is the optimal measurement site, and the CT-FFR value here shows the highest diagnostic performance for myocardial ischemic lesions, especially for moderate stenosis.
4.Effect of measurement site on diagnostic performance of CT-derived fractional flow reserve
Yutao ZHOU ; Na ZHAO ; Yunqiang AN ; Lei SONG ; Chaowei MU ; Jingang CUI ; Tao JIANG ; Li XU ; Hongjie HU ; Lin LI ; Dumin LI ; Wenqiang CHEN ; Lijuan FAN ; Feng ZHANG ; Yang GAO ; Bin LYU
Chinese Journal of Radiology 2025;59(6):704-711
Objective:To investigate the effect of CT-derived fractional flow reserve (CT-FFR) measurement sites on the values and the diagnostic performance, and to determine the optimal measurement site for CT-FFR using invasive FFR as the reference standard.Methods:This study was part of the CT-FFR CHINA clinical trial. Patients with suspected coronary artery disease who were scheduled for invasive coronary angiography (ICA) were prospectively recruited from five clinical centers across the country from November 2018 to March 2020. Each enrolled patient underwent coronary CT angiography (CCTA), CT-FFR, ICA, and invasive pressure wire-based FFR assessments sequentially within one week. Four groups of CT-FFR values were obtained on each enrolled target vessels according to different CT-FFR measurement locations: 1, 2, 3 cm distal to the target lesion, and terminal vessel groups. Spearman and Bland-Altman analyses were used to explore the correlation and consistency of CT-FFR values and FFR values at different measurement sites. The measurement deviation of CT-FFR was also compared. Diagnostic accuracy and performance of CT-FFR, including sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve (AUC), in discriminating myocardial ischemia were analyzed across all measurement site groups on a per-vessel level, using FFR as the reference standard.Results:A total of 289 patients with 345 target lesion vessels were included. According to CCTA, there were 51 target vessels (14.8%) with<50% stenosis, 106 vessels (30.7%) with 50%-69% stenosis, and 188 vessels (54.5%) with stenosis≥70%. At per-vessel level, CT-FFR and FFR values at each measurement position group were highly positively correlated: 1 cm distal to target lesion group, r=0.734 ( P<0.001); 2 cm distal to target lesion group, r=0.732 ( P<0.001); 3 cm distal to target lesion group, r=0.737 ( P<0.001); terminal vessel group was 0.719 ( P<0.001). At per-vessel level, CT-FFR and FFR values of all measurement sites were in good agreement (Bland-Altman analysis results): 1 cm distal to target lesion group, 0.014 (95% LoA 0.002-0.026); 2 cm distal to target lesion group, 0.026 (95% LoA 0.015-0.038); 3 cm distal to target lesion group, 0.040 (95% LoA 0.039-0.051); terminal vessel group, 0.075 (95% LoA 0.064-0.087). And at per-vessel level, the accuracy of diagnosing myocardial ischemia with CT-FFR at 1 cm was highest [84.6% (95% CI 80.4%-88.3%)], and the lowest accuracy in the terminal vessel group [67.0% (95% CI 61.7%-72.0%)]. However, there was no significant difference in the diagnostic accuracy of CT-FFR at 1 cm, 2 cm [80.6% (95% CI 76.1%-84.6%)] and 3 cm [77.5% (95% CI 72.6%-81.7%)]. AUC of CT-FFR at 1 cm distal to the lesion were both highest for global level and moderately stenosis (50%-69%) lesions [0.85 (95% CI 0.81-0.89), 0.84 (95% CI 0.77-0.90)]. And the differences were statistically significant among the four measurement location groups (all P<0.05). Conclusions:The deviation of CT-FFR increases with measurement site distance distal to target lesions. One centimeter distal to the target lesion is the optimal measurement site, and the CT-FFR value here shows the highest diagnostic performance for myocardial ischemic lesions, especially for moderate stenosis.
5.Characteristics of systemic immune microenvironment of DSS-induced acute ulcerative colitis in mice revealed by Mass cytometry
Zongjing LYU ; Jing XUN ; Xiaolin JIANG ; Bin LIU ; Zehan LIU ; Xueliang WU ; Aimin ZHANG ; Yu WU ; Xiangyang YU ; Ximo WANG ; Qi ZHANG
Chinese Journal of Immunology 2025;41(9):2145-2152,中插1
Objective:To explore the characteristics of systemic immune microenvironment during the progression of dextran sulfate sodium(DSS)-induced acute ulcerative colitis(UC)induced in mice by Mass cytometry(CyTOF).Methods:Male C57BL/6 mice were randomly divided into control group and model group.The control group was given normal drinking water for 15 d.The mouse in the model group were given 5%DSS in drinking water,which was changed to normal drinking water after 7 days.In the model group,peripheral blood was collected on days 4,9 and 15,respectively.CyTOF was used to detect the expressions of 33 immune cell markers and changes in cell subsets in peripheral blood of mice,and the characteristics of systemic immune microenvironment in mice with acute UC were analyzed.Results:The cluster analysis of 33 kinds of immune cell markers showed that CD45+cells in peripheral blood of mice with DSS induced acute UC were divided into 23 fine subgroups,among which the proportions of B cell subgroup,T cell subgroup and neutrophil subgroup showed significant changes.A further dimensional reduction cluster analysis of T cell subsets found significant differences in the composition and proportion of the 10 identified T cell subsets.Conclusion:The systemic immune micro-environment map of mice with acute UC induced by DSS has been successfully constructed,and heterogeneity has been found in the systemic immune microenvironment of mice with acute UC.The changes and activation degree of T cell subpopulations are closely re-lated to disease progression and inflammation level.The results of this study provide theoretical basis for assisting the diagnosis,moni-toring the risk,progression,treatment and prognosis of acute UC.
6.Efficacy analysis of a novel inguinal tourniquet for compression hemostasis
Peng-Fei LIU ; Hao SUN ; Meng-Jie DOU ; Ya-Hua LIU ; Shao-Bin CHAI ; Si-Yu CHEN ; Fa-Qin LYU ; Wei CHEN
Medical Journal of Chinese People's Liberation Army 2025;50(6):688-694
Objective To assess the efficacy of a novel inguinal tourniquet in healthy individuals and to investigate the relationship between localized inguinal compression and femoral artery blood flow occlusion.Methods A self-controlled study was conducted.From November 9 to November 30,2024,11 volunteers were recruited at the Third Medical Center of Chinese PLA General Hospital.Three compression methods--finger pressure,a novel groin tourniquet,and a SAM junction tourniquet(SJT)—were applied bilaterally to the inguinal region until distal blood flow signals disappeared.Each compression method was tested in 22 trials with a 5-minute interval between operations.Differences in hemostatic efficacy between bilateral inguinal regions and across compression methods were compared.Subsequently,the novel tourniquet was incrementally pressurized in 120 mmHg multiples using an integrated pressure device to analyze trends in popliteal artery blood flow velocity.Observational indicators included the internal pressure of the tourniquet pressurization device,peak systolic velocity(PSV)of popliteal artery,inguinal surface pressure magnitude,inguinal surface pressure distribution,and pain scores(assessed using a single-dimensional numerical rating scale).Results No statistically significant difference was observed in the minimum pressure required to occlude femoral artery blood flow bilaterally(P>0.05).The success rates of femoral artery blood flow occlusion at the inguinal region were 100%for the novel inguinal tourniquet,SJT,and finger pressure.The novel inguinal tourniquet induced the highest pain scores,ranging from 5 to 8.A significant reduction in PSV of popliteal artery was noted when the intra-tourniquet pressure reached 360 mmHg and 480 mmHg(P<0.05),with a 95%hemostasis efficacy observed within the range of 360-600 mmHg.No significant association was observed between the recovery of popliteal artery blood flow after limb movement and inguinal pressure distribution(P>0.05).The PSV of popliteal artery exhibited the strongest negative correlation with the average pressure within the inguinal compression area(r=-0.79,P<0.001),with a linear regression fitting line of y=69.69-0.13x(P<0.001,R2=0.58).Conclusions The novel inguinal tourniquet effectively occludes femoral artery blood flow within a pressure range of 360-600 mmHg,accompanied by moderate-to-severe pain.Its hemostatic mechanism mainly relies on increasing the mean pressure within the inguinal compression area.
7.Retrospective clinical analysis of 31 cases of necrotizing fasciitis of the neck with or without descending necrotizing mediastinitis.
Bin LI ; Fenglei XU ; Ming XIA ; Xiaoming LI ; Xiaozhi HOU ; Xiaoxu LYU ; Xu GUO
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2025;39(10):971-975
Objective:To summarize the clinical characteristics and management experience of complications in patients with cervical necrotizing fasciitis (CNF) with or without descending necrotizing mediastinitis (DNM), in order to provide a basis for optimizing diagnosis and treatment strategies. Methods:A retrospective analysis was conducted on the clinical data of 31 patients diagnosed with CNF and DNM at Shandong Provincial Hospital Affiliated to Shandong First Medical University between October 2019 and March 2024. A comprehensive evaluation was performed based on the patients' clinical characteristics, metagenomic next-generation sequencing (mNGS) pathogen detection results, imaging assessments, surgical interventions, management approaches for specific complications, and prognostic outcomes. Results:Among the 31 patients, 10 had severe diabetes mellitus. Etiological analysis was summarized as follows: 5 cases were odontogenic, 3 were of tonsillar origin, 3 were due to endogenous esophageal injury, 2 were due to exogenous cervical trauma, 2 originated from a congenital branchial cleft fistula, and 16 cases had an unknown etiology. Among them, 29 patients underwent surgery via an external cervical approach, 1 patient underwent surgery via an intraoral approach, and 1 patient received ultrasound-guided puncture and drainage therapy. Ultimately, 29 patients were cured and discharged (including 1 patient who experienced two instances of major neck vessel rupture and successfully underwent two interventional embolization procedures for hemostasis); 2 patients died after failed rescue efforts due to concurrent sepsis and multiple organ dysfunction. The treatment success rate was 93%, and the mortality rate was 7%. In this cohort of CNF and DNM cases, only a minority had a clearly identified odontogenic cause; although the etiology was unknown in most cases, imaging consistently showed oropharyngeal lymph node necrosis, suggesting a possible pharyngeal origin of infection in adults. The mNGS pathogen profile was predominantly Gram-positive bacteria, accompanied by anaerobic bacilli and fungi. Conclusion:CNF and DNM are severe and rapidly progressive conditions that can lead to life-threatening complications within hours. Timely recognition can reduce unnecessary examinations and expedite treatment.
Humans
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Retrospective Studies
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Fasciitis, Necrotizing/therapy*
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Mediastinitis/complications*
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Neck/pathology*
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Male
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Female
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Middle Aged
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Adult
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Aged
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Prognosis
8.Value of adjuvant chemotherapy in IB-lIA cervical adenocarcinoma: A multicenter retrospective study.
You WU ; Miao AO ; He ZHANG ; Kunyu WANG ; Meixian FANG ; Xueyan LYU ; Guobing CHEN ; Tao LYU ; Bin LI
Chinese Medical Journal 2025;138(17):2192-2194
9.Pathogenic genetic characteristics of Vibrio vulnificus in commercial foods in Pudong New Area of Shanghai
LIANG Yanru ; LYU Bin ; HU Yihuan
China Tropical Medicine 2025;25(1):87-
Objective To understand the contamination status, drug resistance, virulence, genotyping characteristics, and genetic evolution relationships of Vibrio vulnificus in commercially available food in Pudong New Area of Shanghai. Methods The drug susceptibility test was carried out by microbroth dilution method. The whole genome sequencing was used by illumina sequencing platform. The virulence, drug resistance genes and MLST typing were obtained by VFDB, CARD and PubMLST database. Phylogenetic analysis was used by genome-wide single nucleotide polymorphisms. Results The detection rate of Vibrio vulnusis in commercially available food was 4.56% (51/1 119) in 2023, and the dominant detection categories were shellfish(20.16%,25/124), fish(6.74%,19/282), shrimp(6.56%,4/61), ready-to-eat aquatic products(9.09%,2/22) and animal meat(0.33%,1/300). The highest drug resistance rate of Vibrio vulnificus to cefazolin is 23.5%(12/51), followed by ampicillin, tetracycline, and trimethoprim sulfamethoxazole. And 51 strains of Vibrio vulnificus carried 29 kinds of drug resistance genes such as tetracycline(tetA, tetC), quinolones(QnrVC6, QnrVC4), carbapenems(varG), and 163 kinds of virulence genes related to flagella(fla E) and iron ion absorption(hutA). The results of MLST analysis showed that there were 41 ST types(one untyped ), 32 new ST types were generated, and 38 isolates were new ST types, mainly ST326(5.88%,3/51) and ST667(5.88%,3/51). SNP showed that all strains were divided into three branches, and the branches in the phylogenetic tree were longer, and had higher homologous recombination. Conclusion In Pudong New Area, the contamination of Vibrio vulnificus was serious in shellfish, fish, shrimp, ready-to-eat aquatic products and animal meat. The drug resistance spectrum is wide and there are multiple drug resistance phenomena. It carries a variety of virulence genes and drug-resistant genes, and is highly homologous recombination, which has certain food safety risks, and should be strengthened monitoring and management.
10.Analysis of clinical infection characteristics of multidrug-resistant organisms in hospitalized patients in a tertiary sentinel hospital in Shanghai from 2021 to 2023
Qi MAO ; Tenglong ZHAO ; Xihong LYU ; Zhiyuan GU ; Bin CHEN ; Lidi ZHAO ; Xifeng LI ; Xing ZHANG ; Liang TIAN ; Renyi ZHU
Shanghai Journal of Preventive Medicine 2025;37(2):156-159
ObjectiveTo understand the infection characteristics of multidrug-resistant organisms (MDROs) in hospitalized patients in a tertiary sentinel hospital in Shanghai, so as to provide an evidence for the development of targeted prevention and control measures. MethodsData of MDROs strains and corresponding medical records of some hospitalized patients in a hospital in Shanghai from 2021 to 2023 were collected, together with an analysis of the basic information, clinical treatment, underlying diseases and sources of sample collection. ResultsA total of 134 strains of MDROs isolated from hospitalized patients in this hospital were collected from 2021 to 2023 , including 63 strains of methicillin-resistant Staphylococcus aureus (MRSA), 57 strains of carbapenem-resistant Acinetobacter baumannii (CRAB), and 14 strains of carbapenem-resistant Klebsiella pneumoniae (CRKP). Of the 134 strains, 30 strains were found in 2021, 47 strains in 2022 and 57 strains in 2023. The male-to-female ratio of patients was 2.05∶1, with the highest percentage (70.90%) in the age group of 60‒<90 years. The primary diagnosis was mainly respiratory disease, with lung and respiratory tract as the cheif infection sites. There was no statistically significant difference in the distribution of strains between different genders and infection sites (P>0.05). However, the differences in the distribution of strains between different ages and primary diagnosis were statistically significant (P<0.05). Patients who were admitted to the intensive care unit (ICU), had urinary tract intubation, were not artery or vein intubated, were not on a ventilator, were not using immunosuppresants or hormones, and were not applying radiotherapy or chemotherapy were in the majority. There was no statistically significant difference in the distribution of strains for whether received radiotherapy or chemotherapy or not (P>0.05), while the differences in the distribution of strains with ICU admission history, urinary tract intubation, artery or vein intubation, ventilator use, and immunosuppresants or hormones use or not were statistically significant (all P<0.05). The type of specimen was mainly sputum, the hospitalized ward was mainly comprehensive ICU, the sampling time was mainly in the first quarter throughout the year, the number of underlying diseases was mainly between 1 to 2 kinds, the application of antibiotics ≥4 kinds, and those who didn’t receive any surgery recently accounted for the most. There were statistically significant differences in the distribution of strains between different specimen types, wards occupied and history of ICU stay (P<0.05), but no statistically significant difference in the distribution of strains between different sampling times, number of underlying diseases and types of antibiotics applied (P>0.05). ConclusionThe situation of prevention and control on MDROs in this hospital is still serious. Focus should be placed on high-risk factors’ and infection monitoring and preventive measures should be strengthened to reduce the incidence rate of MDROs infection.

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