乙型肝炎病毒荧光PCR基因分型方法的建立和应用

[目的]建立一种在临床上准确、快速、简便地对乙型肝炎病毒进行基因分型（BCD三型）的方法。[方法]大量分析已分型的HBV基因组序列，寻找出HBV各基因型的型特异性碱基的分布规律，设计型特异性的引物和探针，利用已知全序列HBV建立荧光PCR的基因分型方法。并与PCR-RFLP及S基因测序等分型方法作比较。[结果]荧光PCR方法对已知基因组序列的HBV分型结果与基因组分析完全一致，利用大量随机临床标本摸索的最佳反应条件为94℃，1min；94℃，10s，60℃，30s，40个循环。与PCR-RFLP相比，分型结果一致性在84．0％；与S基因测序分型相比，一致性为96．2％。[结论]利用荧光PCR能灵敏、快速、准确地进行乙型肝炎病毒基因分型，方便临床应用和流行病学调查。     

桂林地区乙肝患者HBV核苷类似物耐药位点分析

检测桂林地区乙型肝炎患者体内HBV逆转录酶基因的耐药突变情况,为制订合理的治疗方案提供依据.方法:提取乙肝患者血清中HBV DNA,巢式PCR扩增其逆转录酶基因后测序,Vector NTI Suite 8.0及chromaslite201分析测序结果,Mega4.0进行基因分型.结果:171例患者测序结果中18例发现有基因耐药突变,其中rtA181位点突变7例,占突变总数的38.9％.多药耐药基因突变12例,点突变总数的66.7％.B基因型患者耐药突变率为10.4％,C基因型患者耐药突变率为10.8％,两基因型分布比率的差异无统计学意义(P＞0.05).结论:桂林乙肝患者HBV逆转录酶基因耐药突变组合较为复杂,多药耐药基因突变比率较高.耐药突变的HBV基因型分布比率在所检测患者中无明显差异.     

乙型肝炎病毒基因分型与耐药突变基因位点的检测分析

目的分析乙型肝炎病毒(HBV)基因型及耐药突变基因位点,为指导临床抗病毒治疗合理用药提供依据。方法回顾分析2014年3月～2016年10月安徽中医药大学第一附属医院114例乙型肝炎患者HBV基因分型和拉米夫定(LAM)、替比夫定(LdT)、阿德福韦酯(ADV)和恩替卡韦(ETV)四种核苷类药物(NAs)耐药及耐药突变位点分布情况、HBV核酸(HBVDNA)定量、HBVE抗原(HBeAg)定量、丙氨酸氨基转移酶(ALT)的浓度、血小板(PLT)的含量。结果 114例乙型肝炎患者中检测出HBV基因C型61例,B型45例,D型3例,B+C混合型1例,B+D混合型1例,其他基因型5例。其中C型多于B型(P 0.05)。检出39例NAs耐药,耐药率为34.21%;C型与B型耐药无显著差异。C型患者HBeAg定量高于B型患者(P 0.05),PLT计数低于B型患者(P 0.05)。耐药突变位点最多见于rt204I;C型多位点突变高于B型(P 0.05)。LAM和LdT联合耐药最多见,两种及两种以上多重耐药高于单一耐药(P 0.01)。结论本研究中乙型肝炎患者HBV基因型多为C型,其次为B型。C型患者容易发生肝纤维化,更易发生多位点突变。NAs耐药以LAM和LdT联合耐药为主,多为两种以上联合耐药。检测HBV基因型和耐药突变基因位点对评价乙型肝炎临床治疗效果和指导临床抗病毒治疗合理用药具有十分重要的意义。     

石家庄地区L-核苷类抗病毒药物耐药突变情况分析

目的 分析石家庄地区L-核苷类抗病毒药物耐药突变情况.方法 对石家庄市第五医院就诊的慢性乙型肝炎患者119例进行乙型肝炎病毒逆转录酶区(HBV P区)基因序列测定.结果 119例患者HBV基因分型中B型5例(4.2％),C型114例(95.8％).在B型中4例rtM204I突变;C型中73例rtM204I突变,40例rtM204V突变,1例rtM204I/V突变.119例存在204位点变异的慢性乙型肝炎患者中,共有9种突变模式,其中rtM204I单位点突变占总突变比例的23.5％(28/119),rtM204V单点突变占总突变比例的6％(7/119),rtL180M+rtM204I联合突变占总突变比例的22.7％(27/119),rtL180M+ rtM204V联合突变占总突变比例的27.8％(33/119).结论 慢性乙型肝炎核苷类药物抗病毒治疗过程中,及时监测HBV P区基因耐药情况,尤其是出现rtM204I/V突变后,很容易联合其他位点突变.     

乙型肝炎基因分型同耐药位点突变的关系研究

目的：探讨耐药位点突变与乙型肝炎病毒(HBV)基因分型及其他临床信息的相关性。方法以358例慢性乙型肝炎(CHB)患者为研究对象,采用聚合酶链反应(PCR)-反向点杂交法检测耐药位点及基因分型。收集患者血清 HBV DNA 载量、丙氨酸氨基转移酶(ALT)水平,以及乙型肝炎病毒 e 抗原(HBeAg)、性别、年龄、核苷(酸)类似物用药时间等临床资料。结果所有标本均成功扩增阳性条带。HBV 基因型以 B 型(267例)为主,其次为 C 型(81例)、D 型(10例)。使用核苷(酸)类似物的311患者中完全野生型269例,未用药的47例患者均未见耐药突变。耐药位点突变以204及180/204位点突变为主。耐药突变与患者性别、年龄、血清 HBV DNA 载量、病毒基因型、ALT 水平及 HBeAg 无明显相关性(P >0.05);用药时间越长,耐药突变率越大(P <0.05);180位点突变与204位点突变有一定相关性(P <0.05)。结论PCR-反向点杂交技术可有效检测 HBV 基因型及突变位点,可有效指导临床用药。     

某地区乙型肝炎病毒基因型分布和P区耐药突变模式分析

目的探讨十堰地区乙型肝炎病毒(HBV)基因型分布和P区耐药突变模式及两者的相关性。方法将在该院进行治疗的224例慢性乙型肝炎患者纳入本研究,提取患者血液标本中的HBV DNA,利用ABI 3730型遗传分析仪对DNA进行测序,通过SeqMan软件实施序列比对,分析HBV DNA的基因型和P区耐药突变的情况。结果检出HBV基因型为B型者136例,占60.71%;为C型者62例,占27.68%。二者比例均高于D型、B+C型及未分型者,差异有统计学意义(P0.05)。78例明确出现耐药突变,其中B基因型对于拉米夫定以及阿德福韦的耐药率分别为18.38%与9.56%,与C基因型对于二者的耐药率16.13%与9.68%相比,差异均无统计学意义(P0.05)。B基因型患者M204V、M204I耐药突变的发生率分别为38.60%、17.54%,C基因型患者则分别为28.57%与38.10%,两种基因型患者比较差异无统计学意义(P0.05)。B、C型基因突变患者间YMDD突变(M204V、M204I)及非YMDD突变所占比例比较,差异均无统计学意义(P0.05)。B基因型及C基因型分别与M204V及M204I两种突变均呈正相关。结论十堰地区HBV的基因型主要是B型及C型,发生的HBV耐药突变则以M204I和M204V为主,在临床治疗时应按照基因分型以及耐药突变情况合理选择药物进行慢性乙型肝炎的治疗。     

低水平乙型肝炎病毒表面抗原S区序列分析

目的对低水平乙型肝炎病毒表面抗原进行病毒S区基因分析。方法利用套式PCR法（Nested PCR）特异性扩增乙型肝炎病毒DNA的S区,对PCR扩增产物直接进行DNA序列分析,测序结果经Genotyping软件分型并与美国GeneBank中基因序列进行BLAST比对。结果 37例低水平乙型肝炎病毒表面抗原标本中,成功扩增并测序32份,其余5份扩增产物因不满足测序要求无法进行S区序列分析,32份成功测序标本经Genotyping分型,B型20例,C型12例,分别与GeneBank中收录的AF100309、AB014381同源性最高;血清学分型18例为adr,10例为adw,3例为ayw,1例为ayr,并发现21例S区核酸序列发生点突变,其中15例突变不会引起编码抗原决定簇氨基酸突变,为无义突变,而其他6例点突变均可引起抗原决定族编码的氨基酸的改变,从而导致表面抗原结构改变。结论对低水平乙型肝炎病毒表面抗原患者S区序列研究将为低水平乙型肝炎发病机制、流行病学以及个体化治疗奠定理论基础。     

1株LpsB突变致多黏菌素B耐药鲍曼不动杆菌的全基因测序分析*

摘要:目的研究1 株多黏菌素B高水平耐药的多重耐药鲍曼不动杆菌的全基因组测序的分子特点。方法将1株分离自脑脓肿患者脑脊液标本的多黏茵素高水平耐药鲍曼不动杆菌Z198株作为目标研究菌，通过Nanopore PromethION和llumina No- vaSeq PE150测序平台对其进行全基因组测序,再通过各数据库进行比对分析检测菌株的多位点序列分型(MLST)、抗菌药物耐药基因、毒力基因,同时采用Circos软件对样品基因组组装和注释结果进行可视化分析。Z198基因组上交NCBI数据库。结果全基因组测序发现该菌株含有1 个染色体、1个固有质粒,11个基因岛、4个前噬菌体。通过数据库比对,本菌为ST2型,共携带高达34种抗菌药物耐药基因,主要为IpsB、blaoxA.23、blaox.6、blaxDc-.-3等,同时含有OmpA、磷脂酶C、包膜、溶血素、 生物膜调控系统等19 种主要毒力因子。结论本研究检出 1例LpsB四点突变导致多黏菌素B耐药的鲍曼不动杆菌。该菌株同时携带多重耐药基因及毒力因子。多黏菌素耐药鲍曼不动杆菌外膜脂质修饰的耐药机制及分子流行病学需高度持续关注。     

慢性乙型肝炎患者HBV基因型分析及对6种核苷类药物的耐药性

目的探讨本地区慢性乙型肝炎(CHB)患者乙型肝炎病毒(HBV)基因型分布特点、耐药基因变异及对6种核苷类药物的耐药情况。方法采用实时荧光定量聚合酶链反应(PCR)、巢式PCR和Sanger测序法检测82例CHB患者HBV DNA、HBV耐药基因(HBV P区基因特异性片段)和HBV P区基因耐药相关位点突变,同时检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、乙型肝炎e抗原(HBe Ag)、乙型肝炎e抗体(HBe Ab)。结果 82例CHB患者中检出B型HBV 25例(30.5%)、C型HBV 51例(62.2%)、B+C混合型HBV 5例(6.1%)、未分型1例(1.2%)。B型CHB患者与C型CHB患者之间血清ALT水平、血清AST水平、血清HBV DNA载量及HBeAg、HBeAb阳性率差异均无统计学意义(P0.05)。阿德福韦酯、替诺福韦酯的敏感率最高,均为64.63%。恩替卡韦敏感性下降者占34.15%。替比夫定耐药率为53.66%,恩曲他滨、拉米夫定耐药率均为50.0%。结论本地区CHB患者HBV基因型以C型为主,B型次之。阿德福韦酯、替诺福韦酯的敏感性较高。     

南通地区乙型肝炎病毒P区耐药突变与基因型及其临床意义

目的 探讨南通地区乙型肝炎病毒P区耐药基因突变特征与基因型,为临床合理用药提供科学依据。方法 选择158例经核苷(酸)类似物治疗至少2年以上慢性乙型肝炎(CHB)患者和30例未接受过核苷(酸)类似物治疗的CHB患者作为研究对象,采用PCR产物直接测序法检测HBV P区耐药基因和基因型,同时观察三种主要突变模式与ALT和HBV DNA水平的关系。结果 158例CHB患者检出B基因型42例(26.58%),C基因型116例(73.42%)。131例发生P区不同位点突变,突变率为82.91%。共检出11个HBV突变位点,主要突变位点是M204I,L180M,M204V,A181V和A181T,耐药频率依次为41.14%,37.34%,22.15%,11.39%和10.13%,11个突变位点有21种突变模式。拉米夫定(LAM)耐药相关突变中以L180M和M204V合并出现为主,其次以M204I单独出现; 阿德福韦酯(ADV)耐药相关突变中以A181V为主; 恩替卡韦(ETV)耐药率较低。结论 南通地区HBV基因型以B和C型为主,C型为优势基因型; 耐药突变主要集中在拉米夫定和阿德福韦酯耐药相关的突变位点,而恩替卡韦耐药率较低。多位点耐药突变检测有助于及时发现病毒耐药,更好地指导临床治疗。     

儿童遗传性心律失常的诊治进展

回顾在遗传性心律失常领域最新发表的相关研究,主要关注与儿童心源性猝死关系密切的离子通道病,包括长QT综合征（LQTS）、短QT综合征（SQTS）、Brugada综合征（BrS）和儿茶酚胺敏感性多形性室性心动过速（CPVT）,总结它们在发病机制及诊治方面的进展。     

The acoustic characteristics of the skull

Many investigators have stated that the difficulties of imaging with acoustical energy through the skull result from the marked attenuation of the energy by the skull. In the literature measurements of total attenuation have been confused with those for absorption.Measurements made by us show that absorption by compact bone varies between 2–3 dB cm^?1 MHz^?1 and, in the low megaHertz region appears to be directly proportional to frequency.It has also between shown that the convoluted inner surface of the ivory bone of the inner table of the skull may degrade the collimation and directionality of the beam by refraction.Cancellous bone, such as is present in the dipole of the skull, greatly attenuates the energy. It is postulated that this largely results from scattering. It is also postulated that the energy propagates through cancellous bone as two components, one in the soft tissues and the other partly in the bony spicules. Observations suggest that attenuation due to scattering much more markedly affects the latter of these components and scatters more greatly the higher frequencies in a pulse of broad bandwidth.The energy in each component has varying propagation paths so that the later cycles in the pulse of each component are subject to increasing interference as a result of the variations in propagation times. The two components moreover may have different propagation times so that interference may occur between the pulses of each component as well.All of these phenomena degrade the collimation, coherence, directionality, beam width, pulse length, frequency and other properties of the ultrasonic energy upon which imaging through the skull depends.The interference effects described above are least for the first cycle in the pulse which usually is not the cycle of highest amplitude. Since, in the free field, most of the energy is concentrated around the beam axis, most of the energy in the field which is deflected from its normal propagation path is deflected away from the beam axis. Thus the directionality of the beam is least degraded in the beam axis. The effects of the skull in degrading the properties of the ultrasonic pulse would therefore be lessened if the amplitude of the first cycle of the pulse and the directionality of its energy could be used for imaging.     

Organ Preservation: Current Concepts and New Strategies for the Next Decade

SUMMARY: Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation. This review sets out the history of organ preservation, how applications of hypothermia have become central to the process, and what the current status is for the range of solid organs commonly transplanted. The science of organ preservation is constantly being updated with new knowledge and ideas, and the review also discusses what innovations are coming close to clinical reality to meet the growing demands for high quality organs in transplantation over the next few years.     

2017年呼吸疾病主要进展

2017年,国内外学者在呼吸系统疾病的临床和基础领域均进行了深入研究,不仅对相关指南进行了更新,并且针对一些临床热点、难点问题达成专家共识,现就2017年呼吸疾病相关进展作一简单介绍。     

家族性噬血细胞性淋巴组织细胞增生症1例并文献复习

目的加强对家族性噬血细胞性淋巴组织细胞增生症（familially hemophagocytic lymphohistiocytosis,FHL）的认识。方法报道确诊为FHL的新病例1例,结合国内外报道的FHL的病例,对该病的临床特点进行汇总分析。结果FHL2常与PRF1基因突变相关,约20%~40%的患者存在穿孔素基因突变。结论对于有阳性家族史,基因诊断明确,应尽早行化疗或者造血干细胞移植。若无家族史,未发现与继发性HLH相关的原发病因,可考虑行基因筛查以明确是否存在FHL的可能。     

COVID‐19 and COVID‐19 vaccine‐related dermatological reactions: An interesting case series with a narrative review of the potential critical and non‐critical mucocutaneous adverse effects related to virus,therapy, and the vaccination

This narrative review article was conducted to lay out a summarized but exhaustive review of current literature over mucocutaneous manifestations in 4 dimensions of SARS‐CoV‐2 pandemic: virus itself, treatment‐related, vaccine‐induced, and alteration of chronic dermatologic diseases following infection. Virus and vaccine‐related were mainly self‐limited and non‐severe. Treatment‐related reactions could be life‐threatening.     

Burkitt's lymphoma of maxillary gingiva: A case report

Burkitt's lymphoma(BL) is an aggressive form of nonHodgkin's B-cell lymphoma with three variants namely endemic, sporadic, and immunodeficiency-associated types. It is endemic in Africa and sporadic in other parts of the world. While the endemic form is widely reported to occur in early childhood and commonly involves the jaw bones, the sporadic form typically presents as an abdominal mass. This presentation reports a rare case of sporadic form of BL clinically manifesting as a generalized gingival enlargement in an immunocompetent adult male which demonstrated an aggressive behavior. The patient reported with a prominent anterior gingival swelling of 6 mo duration which slowly enlarged in size and associated with multiple lymph node involvement. Microscopic examination of the lesion using H, E and immunohistochemical diagnosis confirmed the diagnosis as BL. The patient succumbed to the disease before any therapy could be instituted. Since a wide array of causes can be attributed to gingival enlargements, it is necessary to consider malignancies as one of the important differential diagnosis so as to facilitate the need for appropriate diagnosis and prompt treatment.     

Real‐life effectiveness of biological therapies on symptoms in severe asthma with comorbid CRSwNP

BackgroundWe aimed to evaluate the effectiveness of different antibody therapies on nasal polyp symptoms in patients treated for severe asthma.MethodsWe performed a retrospective analysis of patients with severe asthma and comorbid CRSwNP who were treated with anti‐IgE, anti‐IL‐5/R or anti‐IL‐4R. CRSwNP symptom burden was evaluated before and after 6 months of therapy.ResultsFifty patients were included hereof treated with anti‐IgE: 9, anti‐IL‐5/R: 26 and anti‐IL‐4R: 15 patients. At baseline median SNOT‐20 was similar among groups (anti‐IgE: 55, anti‐IL‐5/R: 52 and anti‐IL‐4R: 56, p = 0.76), median visual analogue scale (VAS) for nasal symptoms was 4, 7 and 8 (p = 0.14) and VAS for total symptoms was higher in the anti‐IL‐4R group (4, 5 and 8, p = 0.002). After 6 months SNOT‐20 improved significantly in all patient groups with median improvement of anti‐IgE: −8 (p < 0.01), anti‐IL‐5/R: −13 (p < 0.001) and anti‐IL‐4R: −18 (p < 0.001), with larger improvement in the anti‐IL‐4R group than in anti‐IgE (p < 0.001) and anti‐IL‐5/R (p < 0.001) groups. VAS nasal symptoms improved by median anti‐IgE: 0 (n.s.), anti‐IL‐5/R: −1 (p < 0.01) and anti‐IL‐4R: −3 (p < 0.001), VAS total symptoms by anti‐IgE: −1 (n.s.), anti‐IL‐5/R: −2 (p < 0.001) and anti‐IL‐4R: −2 (p < 0.001).ConclusionsTreatment by all antibodies showed effectiveness in reducing symptoms of CRSwNP in patients with severe asthma, with the largest reduction observed in anti‐IL‐4R‐treated patients.     

动态心电图临床操作标准化方法

动态心电图,又称Holter或Holter检查,是一种评价各种心脏病患者心电图异常的简便、高效、准确、安全的无创检查,广泛用于心律失常的相关症状评价,心肌缺血的诊断,心脏病患者的预后和日常生活能力评估,药物疗效评价,起搏器等埋藏式心脏电治疗装置监测等领域。目前动态心电图已广泛用于于临床各级医疗机构,为了更好地发挥其作用,有必要对该项技术进行规范化培训。本文参考相关指南、共识及专家建议,结合作者经验,撰写动态心电图临床操作标准化方法供临床使用时参考。