Objective:Based on the principle that the aggregation-induced emission (AIE) fluorescent probe 6PD-DPAN could bind and aggregate with bacteria, and the fluorescence intensity could reflect the quantity of bacteria, a new method for rapid, convenient, and accurate bacterial drug sensitivity testing was established, which provided a basis for rapid and accurate clinical drug use.Methods:This was a methodological evaluation study. A total of 107 clinical isolates were collected from Houjie Hospital of Dongguan City from January to December 2022, among which 46 isolates were used for the establishment of the new method, and 61 isolates were used for methodological validation. The minimum inhibitory concentration (MIC) determined by broth microdilution method was used as the gold standard, and three antibacterial drugs, gentamicin, levofloxacin, and cefotaxime, were used as experimental drugs. The AIE plate was incubated for 4 hours, and the fluorescence intensity was measured every half an hour to draw a fluorescence change curve. The MIC results were compared with the CLSI breakpoints to determine the bacteria as sensitive, intermediate, or resistant. To simplify the detection process, the ratio of fluorescence intensity at 4 hours(R) was calculated, and the ROC curve was used to analyze the efficacy of R in determining bacterial growth and establish its cutoff value. The new method was used to determine the MIC of 61 clinical isolates, with broth microdilution method as the gold standard. The basic consistency, categorical consistency, very major errors, and major errors of the new method were analyzed, and the consistency between the two methods was determined by the Kappa test.Results:ROC curve analysis of the R after 4 hours of culture: The cut-off value was 3.0, with both sensitivity and specificity for determining bacterial growth being 100%. The median (interquartile) R for bacterial growth inhibition was 11.1 (8.6, 14.4); the median R-value for bacterial growth was 1.1 (1.0, 1.2). Compared to the gold standard, the newly established method showed 100% (61/61) essential agreement in detecting MICs of 61 clinical isolates, with a categorical agreement of 96.7% (59/61). There were no very major or major errors, and the Kappa value was 0.94, indicating good consistency between the newly established method and the microbroth dilution method.Conclusions:This study successfully established a new method for bacterial drug sensitivity testing based on AIE technology, which could obtain satisfactory results within 5 hours, providing a basis for early precision drug treatment in clinical practice.

Introduction: Circular RNA (circRNAs) are a type of non-coding RNA (ncRNAs) with a wealth of functions. Recently, circRNAs have been identified as important regulators of diabetic kidney disease (DKD), owing to their stability and enrichment in exosomes. However, the role of circRNAs in exosomes of tubular epithelial cells in DKD development has not been fully elucidated. Methods: In our study, microarray technology was used to analyze circRNA expression in cell supernatant exosomes isolated from HK-2 cells with or without high glucose (HG) treatment. The small interfering RNAs (siRNA) and plasmid overexpression were used to validate functions of differentially expressed circRNAs. Results: We found that exosome concentration was higher in HG-stimulated HK-2 cells than in controls. A total of 235 circRNAs were significantly increased and 458 circRNAs were significantly decreased in the exosomes of the HG group. In parallel with the microarray data, the qPCR results showed that the expression of circ_0009885, circ_0043753, and circ_0011760 increased, and the expression of circ_0032872, circ_0004716, and circ_0009445 decreased in the HG group. Rescue experiments showed that the effects of high glucose on regulation of CCL2, IL6, fibronetin, n cadherin, e cadherin and epcam expression can be reversed by inhibiting or overexpressing these circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses indicated that circRNA parental genes are associated with glucose metabolism, lipid metabolism, and inflammatory processes, which are important in DKD development. Further analysis of circRNA/miRNA interactions indicated that 152 differentially expressed circRNAs with fold change (FC) >= 1.5 could be paired with 43 differentially expressed miRNAs, which are associated with diabetes or DKD. Discussion: Our results indicate that exosomal circRNAs may be promising diagnostic and therapeutic biomarkers, and may play a critical role in the progression of DKD.

目的 探讨尿外泌体中致纤维化基因表达在诊断早期糖尿病肾病的应用价值.方法 选取2019年5月—2020年6月于广东医科大学附属厚街人民医院住院的新诊断早期2型糖尿病肾病患者16例及2型糖尿病肾病患者16例,将32例患者分别纳入研究组A组(早期2型糖尿病肾病组)和B组(2型糖尿病肾病组).同时选取同期到医院进行体检的健康体检者16名,纳入对照组.对高糖刺激肾小管上皮外泌体miRNA进行系统分析(芯片检测).收集了两组DN患者肾穿刺标本,进行组化染色,对比两组患者血肌酐、尿白蛋白、尿钠等的水平以及对比两组患者尿外泌体中转化生长因子-β1(TGF-β1)和结缔组织生长因子(CTGF)的mRNA的表达等相关情况.结果 研究组血肌酐、尿白蛋白、尿钠的水平明显高于对照组,差异有统计学意义(P<0.05);研究组A组的血肌酐、尿白蛋白、尿钠的水平低于研究组B组,差异有统计学意义(P<0.05).研究组A组和B组患者尿液中的TGF-β1和CTGF的mRNA表达水平均明显高于对照组(2.2±0.8)、(4.1±1.2)vs(1.0±0.2)、(1.8±1.1)、(3.8±1.8)vs(1.1±0.3),差异有统计学意义(P<0.05).研究B组患者尿液中的TGF-β1和CTGF的mRNA表达水平明显高于研究A组,分别为(4.1±1.2)vs(2.2±0.8)和(3.8±1.8)vs(1.8±1.1),差异有统计学意义(P<0.05).结论 糖尿病肾病患者尿外泌体中致纤维化基因TGF-β1和CTGF可能是临床诊断早期糖尿病肾病及其进展的重要辅助生物学标志物.

目的 构建一种基于深度学习的革兰染色图像自动分类模型EKNet,实现对泌尿系统感染常见的大肠埃希菌(Eco)和肺炎克雷伯菌(Kpn)的快速诊断.方法 将预处理后的细菌图像输入残差网络模型,计算交叉熵损失,通过反向传播算法进行参数优化,获得预训练模型EKNet.共采集368例Eco和292例Kpn显微图像,其中272例Eco和214例Kpn进行训练,40例Eco和30例Kpn进行验证,余下104例图像进行测试.以ResNet50模型和AlexNet模型作为对照,评估EKNet模型的实用性.结果 EKNet模型测试一个样本的平均用时是0.165 s,优于ResNet50模型的0.38 s和AlexNet模型的0.66 s;图像识别准确率达98.8％,优于ResNet50模型的96.4％和AlexNet模型的78.6％.EKNet模型对104例临床测试的显微图像输出结果与尿培养的结果完全吻合,正确率为100％.结论 成功构建Eco和Kpn的图像诊断模型EKNet,为临床泌尿系统感染的快速诊断提供参考.

目的 检测妊娠糖尿病(GDM)患者血清中胰岛素受体底物-1(IRS-1)的表达水平,同时分析其与胰岛素抵抗(IR)的相关性.方法 选择该院2019年5月—2020年5月收治的分娩期孕妇86例,根据OGTT试验结果分为GDM组44例和正常糖耐量组(NGT组)42例,使用ELISA的方法检测两组患者血清IRS-1的表达水平,同时检测患者空腹血糖(FPG)、餐后1 h血糖(1 hPG)、餐后2 h血糖(2 hPG)、空腹胰岛素(FINS)、空腹C肽、胰岛素抵抗指数(HOMA-IR)、糖化血红蛋白(HbA1c)指标.结果 GDM组患者FPG、1 hPG、2 hPG、FINS、空腹C肽、HOMA-IR、HbA1c水平均高于NGT组,而IRS-1水平低于NGT组,差异有统计学意义(P<0.05).同时经过Pearson相关性分析显示,GDM组患者IRS-1表达水平与患者HOMA-IR、FPG、1 hPG、2 hPG、FINS、HbA1c水平呈明显负相关(P<0.05).结论 GDM孕妇存在明显的IR,其与患者血清IRS-1的表达呈负相关,因此低IRS-1在GDM的发生发展中起到一定的作用,这一结果为将来GDM的治疗提供了依据.

目的 建立一种可同时、快速检测2型糖尿病患者降糖药相关药物基因多态性的液相芯片检测系统.方法 根据7个目的基因的rs号,在Genbank中查找其靶位点附近碱基序列(目的基因包括磺脲类受体1,转录因子7类似物2,胰岛素受体底物1,过氧化物酶体增殖物激活受体γ,有机阳离子转运蛋白与多药和有毒化合物排出家族,有机阴离子转运蛋白家族成员1B1等),以PrimerPlex软件设计等位基因特异性引物,Primer6.0软件设计含检测位点的PCR引物,通过多重PCR扩增,等位基因特异性引物延伸(ASPE),MagPlex～Tag微球杂交,液相芯片系统Luminex 200检测荧光信号,确定基因型,优化反应体系并以5份代表性标本进行方法学评价.收集2019年1月～12月东莞市厚街医院新诊2型糖尿病患者血液样本115例,采用建立的系统检测上述7个靶位点,并随机选取25例与测序结果比较.结果 7个目标基因经PCR扩增,产物电泳成像后清楚可见7条目标条带,无非特异性条带;经优化ASPE杂交条件,选择退火温度55℃,Biotion～dCTP浓度与dCTP浓度的比值为3:1,37℃孵育45 min时检测效果最佳;临床样本检测结果显示:纯合子荧光强度中位值(median fluorescence intensity,MFI)比率均>0.9或<0.1,杂合子MFI比率在0.4～0.6之间.纯合子MFI比率批内批间CV在0.9％～3.3％和2.1％～4.6％,而杂合子则在2.9％～7.3％和5.2％～11.2％;DNA最低检测限为0.75ng;25例样本的检测结果与测序结果完全一致,准确度100％.结论 该研究成功建立了一种新的液相芯片检测系统,高效便捷,能同时检测7个目的基因型,满足临床的需求.

目的 了解2019年该院临床分离细菌对抗菌药物的耐药性.方法 收集2019年该院临床分离的1220株非重复菌株,采用纸片扩散法或自动化仪器法进行药敏试验,按相关标准判读药敏结果,采用WHONET5.6软件进行数据分析.结果 1220株细菌中,革兰阴性菌占72.1％,革兰阳性菌占27.9％.较常见的5种细菌分别为大肠埃希菌(34.1％)、肺炎克雷伯菌(10.7％)、金黄色葡萄球菌(10.2％)、铜绿假单胞菌(7.0％)和凝固酶阴性葡萄球菌(6.2％).耐甲氧西林金黄色葡萄球菌(MRSA)和耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)的检出率分别为15.2％ 和65.8％.MRSA和MRCNS对各类抗菌药物的耐药率明显高于甲氧西林敏感金黄色葡萄球菌(MSSA)和甲氧西林敏感凝固酶阴性葡萄球菌(MSCNS).未发现对万古霉素耐药的葡萄球菌.链球菌属细菌对红霉素、克林霉素耐药率高,未发现对万古霉素、青霉素耐药的菌株.产超广谱β-内酰胺酶的大肠埃希菌、肺炎克雷伯菌检出率分别为27.9％ 和26.9％.肠杆菌科细菌对碳青霉烯类抗菌药物最敏感,未发现耐药菌株.铜绿假单胞菌对亚胺培南和美罗培南的耐药率分别为8.4％ 和12.2％,对头孢吡肟的耐药率最低(0.0％).结论 细菌耐药性具有地域特点,定期进行细菌耐药性监测有助于了解该院细菌耐药性的变迁,为临床经验用药和合理用药提供依据.