四种实验室检测技术对利福平和异烟肼耐药性检测的临床价值

目的 探讨微孔板法、实时荧光PCR熔解曲线技术(简称“熔解曲线法”)、多色巢式实时荧光定量PCR技术(简称“Xpert 法”)和罗氏药物敏感性试验(L-J药敏试验,简称“比例法”)用于快速筛查耐多药结核病(MDR-TB)的临床价值。方法 从医院信息系统(hospital information system,HIS)连续收集2014年7月至2018年3月重庆市公共卫生医疗救治中心收治的确诊为结核病,且具有微孔板法、比例法、熔解曲线法、Xpert 法检测MTB对利福平、异烟肼耐药性诊断结果的2792例患者资料;其中微孔板法、比例法采用阳性分离菌株检测,熔解曲线法和Xpert 法采用患者标本直接检测。纳入同时具有微孔板法和比例法耐药性检测结果的1488例患者作为研究对象,其中341例行微孔板法+比例法+Xpert法检测利福平耐药性,87例行微孔板法+比例法+熔解曲线法检测利福平耐药性,66例行微孔板法+比例法+熔解曲线法检测异烟肼耐药性。以比例法为标准,采用SPSS 13.0软件分别计算微孔板法、熔解曲线法、Xpert法检测利福平和(或)异烟肼耐药性的敏感度、特异度、符合率、Kappa值等。结果 以比例法为标准,微孔板法、Xpert法、熔解曲线法检测利福平耐药性的敏感度、特异度、阳性预测值、阴性预测值、符合率、Kappa值分别为97.2%(731/752)、96.9%(713/736)、96.9%(731/754)、97.1%(713/734)、97.0%(1444/1488)、0.94,97.2%(140/144)、94.9%(187/197)、93.3%(140/150)、97.9%(187/191)、95.9%(327/341)、0.92,97.1%(33/34)、84.9%(45/53)、80.5%(33/41)、97.8%(45/46)、89.7%(78/87)、0.79;微孔板法和熔解曲线法检测异烟肼耐药性的敏感度、特异度、阳性预测值、阴性预测值、符合率、Kappa值分别为94.8%(751/792)、95.7%(667/697)、96.3%(751/780)、94.2%(667/708)、97.9%(1418/1448)、0.91, 97.3%(36/37)、86.2%(25/29)、90.0%(36/40)、96.2%(25/26)、92.4%(61/66)、0.84。结论 微孔板法、熔解曲线法、Xpert 法检测利福平和(或)异烟肼耐药性均具有较高的敏感度和特异度,适合快速筛查耐多药结核病;微孔板法还能获得各药物最低药物浓度,为临床用药剂量的选择提供参考依据。     

Persistent inhibition of Candida albicans biofilm and hyphae growth on titanium by graphene nanocoating

ObjectivesCandida albicanscolonizes biomaterial surfaces and are highly resistant to therapeutics. Graphene nanocoating on titanium compromises initial biofilm formation. However, its sustained antibiofilm potential is unknown. The objective of this study was to investigate the potential of graphene nanocoating to decrease long-term fungal biofilm development and hyphae growth on titanium.MethodsGraphene nanocoating was deposited twice (TiGD) or five times (TiGV) on grade 4 titanium with vacuum assisted technique and characterized with Raman spectroscopy and atomic force microscope. The biofilm formation and hyphae growth of C. albicans was monitored for seven days by CFU, XTT, confocal, mean cell density and scanning electronic microscopy (SEM). Uncoated titanium was the Control. All tests had three independent biological samples and were performed in independent triplicates. Data was analyzed with one- or two-way ANOVA and Tukey's HSD (α = 0.05).ResultsBoth TiGD and TiGV presented less biofilms at all times points compared with Control. The confocal and SEM images revealed few adhered cells on graphene coated samples, absence of hyphae and no features of a mature biofilm architecture. The increase in number of layers of graphene nanocoating did not improve its antibiofilm potential.SignificanceThe graphene nanocoating exerted a long-term persistent inhibitory effect on the biofilm formation on titanium. The fewer cells that were able to attach on graphene coated titanium were scattered and unable to form a mature biofilm with hyphae elements. The findings open opportunities to prevent microbial attachment and proliferation on implantable materials without the use of antibiotics.     

Genetic Analysis and Attribution of Microbial Forensics Evidence

Because of the availability of pathogenic microorganisms and the relatively low cost of preparing and disseminating bioweapons, there is a continuing threat of biocrime and bioterrorism. Thus, enhanced capabilities are needed that enable the full and robust forensic exploitation and interpretation of microbial evidence from acts of bioterrorism or biocrimes. To respond to the need, greater resources and efforts are being applied to the burgeoning field of microbial forensics. Microbial forensics focuses on the characterization, analysis and interpretation of evidence for attributional purposes from a bioterrorism act, biocrime, hoax or inadvertent agent release. To enhance attribution capabilities, a major component of microbial forensics is the analysis of nucleic acids to associate or eliminate putative samples. The degree that attribution can be addressed depends on the context of the case, the available knowledge of the genetics, phylogeny, and ecology of the target microorganism, and technologies applied. The types of genetic markers and features that can impact statistical inferences of microbial forensic evidence include: single nucleotide polymorphisms, repetitive sequences, insertions and deletions, mobile elements, pathogenicity islands, virulence and resistance genes, house keeping genes, structural genes, whole genome sequences, asexual and sexual reproduction, horizontal gene transfer, conjugation, transduction, lysogeny, gene conversion, recombination, gene duplication, rearrangements, and mutational hotspots. Nucleic acid based typing technologies include: PCR, real-time PCR, MLST, MLVA, whole genome sequencing, and microarrays.     

代谢产物在检测阿片类物质滥用中的研究进展

目的对近年来检测阿片类物质滥用的代谢产物进行综述,为加强阿片类物质的管理、确证滥用导致的死亡等提供依据,也可以用于区别滥用和正常摄入。方法通过大量检索检测阿片类物质滥用的代谢产物相关资料,总结归纳阿片类物质体内代谢过程、常用代谢产物检测的优缺点及应用。结果海洛因、含可待因的药物和罂粟类物质等均能代谢生成吗啡,导致假阳性结果。目前尚未有明确统一的金标准用于准确判断阿片类物质的滥用,滥用确证时除了检测常用的代谢产物指标6-单乙酰吗啡和吗啡外,乙酰可待因、可待因、吗啡与可待因浓度比值、蒂巴因与罂粟碱及那可汀的代谢物也可附加检测。结论在确证阿片类物质滥用时,需谨慎判断代谢产物检测结果的可靠性,并详细询问被检测者近期的服药史。     

三种西药制剂微生物限度检查方法适用性试验的研究

目的建立复方氨酚烷胺胶囊、复方盐酸普鲁卡因胶囊和诺氟沙星胶囊三种制剂的微生物限度检查方法。方法按照2015年版《中国药典》四部的规定测定三种制剂对5个试验菌株的回收比值,并进行控制菌检查的方法适用性试验。结果复方氨酚烷胺胶囊与复方盐酸普鲁卡因胶囊均采用稀释法(1∶100)+薄膜过滤法(500mL/膜)进行需氧菌总数的测定,诺氟沙星胶囊采用稀释法(1∶100)+中和剂法+薄膜过滤法(500mL/膜)进行需氧菌总数的测定;三种制剂均采用平皿法(1∶10)进行霉菌和酵母菌总数的测定;复方氨酚烷胺胶囊采用薄膜过滤法(1∶20,冲洗500mL)、复方盐酸普鲁卡因胶囊采用常规法、诺氟沙星胶囊采用中和剂法+薄膜过滤法(1∶20,冲洗800mL)进行控制菌大肠埃希菌的检查。结论上述方法可作为三种制剂的微生物限度检查方法。     

16S rDNA序列分析法在注射剂微生物污染鉴定及溯源分析中的应用

目的 对一次注射剂无菌检查阳性结果进行溯源。方法 采用16S rDNA序列分析法对污染菌A1和污染调查中采集的16株葡萄球菌进行鉴定和同源性分析,并将分析结果用脉冲场凝胶电泳进行验证。结果 A1的鉴定结果为科氏葡萄球菌;科氏葡萄球菌科氏亚种菌株的核糖体16S亚基序列完全相同;结合PFGE分析显示A1来自无菌检查使用的隔离器污染。结论 制药企业可以使用16S rDNA序列分析法进行生产和检验中污染菌的鉴定和溯源,但对于与如科氏葡萄球菌科氏亚种一样核糖体16S亚基序列完全相同的菌株,需要结合其他同源性分析方法进行最终的判定。     

一株红树内生真菌Penicillium sp. JY246次级代谢产物及其活性研究

目的 研究一株红树角果木来源内生真菌Penicillium sp. JY246的次级代谢产物及其生物活性。方法 利用硅胶柱层析、Sephadex LH-20凝胶柱层析、半制备HPLC等方法,对该菌发酵产物的乙酸乙酯浸膏进行分离纯化;利用NMR、MS等波谱解析方法以及与文献数据对照,鉴定化合物的结构;通过抗菌和抗虫活性模型对化合物的生物活性进行评价。结果 从内生真菌Penicillium sp. JY246中分离得到12个化合物,分别为7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (1),(2′S)-2-(propan-2′-ol)-5-hydroxy-benzopyran-4-one (2),2, 3-dihydro-5-hydroxy-2(S)-methyl-4H-1-benzopyran-4-one (3),(11S)-diaprothin (4),questin (5),4-hydroxy-3-prenylbenzoic acid (6),4-methoxy-6-styryl-pyran-2-one (7),(R) 4-hydroxy-2-oxo-1-pyrrolidineacetamide (8),p-hydroxy-benzaldehyde (9),4-hydroxyacetophenone (10),apocynin (11)和1-(2,6-dihydroxyphenyl) ethan-1-one (12)。结论 从内生真菌Penicillium sp. JY246的次级代谢产物中分离得到12个单体化合物,并对所有化合物进行抗菌和抗虫活性测试。结果表明,化合物1, 2, 4和5显示抗细菌活性;化合物4, 5和8对棉铃虫幼虫显示了生长抑制活性。     

急性坏死性胰腺炎合并感染的病原菌及耐药性分析

目的：研究急性坏死性胰腺炎（ANP）合并感染的病原菌分布及耐药性,并探讨其对ANP患者预后的影响。 方法：回顾性分析2010年10月—2014年10月收治的72例有明确病原学依据的ANP合并感染患者的临床资料。 结果： 72例ANP患者中,腹腔及腹膜后感染47例（65.28%）,呼吸道感染37例（51.39%）,血流感染32例（44.44%）,其中血流感染与患者死亡密切相关（P<0.05）。检出病原菌235株,其中革兰阴性细菌159株（67.66%）,革兰阳性细菌60株（25.53%）,真菌16株（6.81%）。获得的病原菌中排名前6位的依次是：鲍曼不动杆菌（24.68%）,铜绿假单胞菌（8.94%）,肺炎克雷伯菌（8.09%）,屎肠球菌/粪肠球菌（8.09%）,大肠埃希菌（7.66%）,金黄色葡萄球菌（4.68%）。耐药性分析显示,鲍曼不动杆菌和铜绿假单胞菌对亚胺培南耐药率分别达95.92%和52.63%,对头孢哌酮/舒巴坦的耐药率分别为59.26%和50.00%。肺炎克雷伯菌和大肠埃希菌产超广谱β-内酰胺酶（ESBLs）检出率分别为64.29%和80.00%,对头孢哌酮/舒巴坦的耐药率分别为31.58%和18.75%,而对亚胺培南的耐药率分别为23.08%和7.14%。19株屎肠球菌/粪肠球菌中仅1株对万古霉素耐药,对利奈唑烷尚未发现耐药菌株。耐甲氧西林金黄色葡萄球菌检出率为72.73%,对万古霉素、利奈唑烷和呋喃妥因尚未发现耐药菌株。16株真菌对常用抗真菌药物均未发现耐药菌株。 结论：血流感染是ANP患者死亡的重要原因。ANP患者的病原菌仍以革兰阴性菌为主,但革兰阳性菌和真菌的比例不容忽视。多重耐药菌已成为ANP合并感染的巨大的挑战。     

Experiencia en práctica clínica de optimización de tiopurinas mediante determinación de sus metabolitos en la enfermedad inflamatoria intestinal

Introduction

Thiopurine therapy can be optimised by determining the concentration of the drug's metabolites.