等位基因特异性PCR检测HBV G1896A变异的缺陷和改进

目的：探讨AS-PCR检测HBV DNA前C区点突变(G1896A)的缺陷和改进方法。方法：采用单管双复性温度方案进行AS-PCR检测。以不同浓度的HBV野生型和G1896A突变型重组质粒DNA为模板和不同扩增条件分析AS-PCR的缺陷。收集100例HBsAg阳性／HBeAg阴性患者和60例HBsAg阳性／HBeAg阳性急者的血清,根据荧光定量PCR的检测结果,调整血清中的HBV DNA合量,采用AS-PCR比较检测调整前后的差异,探讨AS-PCR的改进办法。结果：AS-PCR检测质粒DNA时显示,野生模板数量较大和特异性扩增循环次数过多时易出现假阳性,而特异性扩增循环次数过少时检测的敏感性下降。在检测血清标本时,HBeAg阳性患者和血清HBV DNA含量高的HBeAg阴性患者易出现假阳性。调整HBV DNA含量后,AS-PCR的这种假阳性可以消除。改进AS-PCR检测血清标本显示,在HBeAg阴性患者中,血清HBV DNA含量高的G1896A变异检出率显著低于HBV DNA含量较低患者(P＝0．0192)。结论：AS-PCR存在其特异性受野生型原始模板数量影响的缺陷,不适用于检测原始模板数量未知、非均一、且波动较大的临床标本。调整标本中的原始模板数量可提高AS-PCR检测慢性乙型肝炎患者血清中HBV G1896A变异的特异性。G1896A变异不是HBsAg阳性／HBeAg阴性患者出现HBV高复制的主要发生机制。     

辽宁省乙肝病毒免疫学标志物与DNA含量检测的分析

目的 探讨乙肝病毒感染免疫学标志物常见模式与HBV DNA含量检测之间的关系.方法 收集225例辽宁省乙型肝炎病毒感染者及20例健康对照者血清,采用ELISA方法进行乙型肝炎病毒免疫学标志物检测,并采用荧光定量PCR方法检测其HBV DNA含量.结果 各组之间DNA含量有差异(P＜0.001).大三阳组患者血清HBV DNA阳性检出率明显高于除HBsAg( )、HBeAb( )、HBcAb-IgM( )组(P=0.232)以外的其他各组(P＜0.001).HBeAg阳性和HBV DNA阳性有较高的相关性(P＜0.001).各组之间ALT含量无差异,而ALP、GGT均存在差异(P＜0.05).结论 辽宁地区各模式分组中大三阳组患者HBV DNA的阳性检出率及HBV DNA的平均含量最高,HBeAg检测和DNA定量检测两者呈正相关,HBV DNA含量与肝功能的损坏程度可能相关.     

基于CRISPR-Cas13a技术的乙型肝炎病毒ccDNA检测方法临床应用评价

摘要:目的评价基于CRISPR-Cas13a技术的乙型肝炎病毒(HBV)共价闭合环状DNA(ceDNA)检测方法的应用价值。方法收集25例HBV感染患者和5例非HBV感染患者的肝组织标本,提取肝组织的总DNA,依次使用HindII内切酶和质粒安全 性ATP依赖DNA酶(PSAD)消化;设计HBV cceDNA的特异性引物,扩增消化后的产物;利用CRISPR/Cas13a技术建立CRISPR-HBV cceDNA检测方法,通过HBV cceDNA质粒和临床样本对该方法进行评价,并利用数字PCR和荧光定量PCR方 法进行比较。结果本研究建 立的方法检测HBV cccDNA质粒的灵敏度为1 copies/μL,显著高于数字PCR( 10 opies/μL,P<0.01)和荧光定量PCR( 10'' copies/uL,P<0.01);建立的方法检测HBV cceDNA阳性样本的灵敏度为1 copies/uL,与数字PCR 相同,高于荧光定量PCR( 10 copies/ μL,P<0.01);建立的方法对25例HBV感染的临床肝组织样本的检出阳性率为80%,均高于数字PCR(64%)和荧光定量PCR(48%)。结论本研究 建立的CRISPR-HBV cceDNA检测方法在乙肝患者肝组织标本 HBVcccDNA检测中具有较高的灵敏度,为评价乙肝患者的临床治疗效果和监测停药复发等情况提供了有效帮助。     

HBsAg阳性卵巢肿瘤病人卵巢组织HBV DNA检测

目的 通过检测乙型肝炎病毒（HBV）表面抗原（HBsAg）阳性卵巢肿瘤病人卵巢组织中HBV DNA,探讨HBV可否感染人类卵巢组织细胞,为HBV经生殖细胞垂直传递感染提供实验依据。方法 自20例HBsAg阳性卵巢肿瘤病人卵巢组织蜡块中提取DNA,应用聚合酶链反应（PCR）和荧光原位杂交（FISH）方法 检测卵巢肿瘤病人卵巢组织细胞中HBV DNA。以20例HBsAg阴性卵巢肿瘤病人卵巢石蜡组织作为对照。结果 HBsAg阳性卵巢肿瘤病人卵巢组织存在HBV DNA,与对照组比较,差异有极显著意义（χ^2=24.000、21.539,P〈0.01）。结论 HBV可以感染卵巢组织细胞,HBV有可能通过卵细胞进行垂直传播。     

HBV血清标记物不同组合模式与荧光定量结果之间的关系

目的：对HBV血清标记物不同组合模式与HBVDNA定量结果进行对比分析。方法：采用ELISA方法检测乙肝病毒血清标记物，荧光定量PCR检测HBV—DNA，比较二者关系。结果：HBsAg、HBeAg、HBcAb阳性组、HB—sAg、HBeAg阳性组及HBsAg、HBsAb、HBeAg、HBcAb阳性组HBV—DNA检出率和平均水平含量均较高。结论：荧光定量PCR检测HBV—DNA能准确反映体内HBV真实感染和复制情况，同时进行HBV血清标记物的检测与HBV—DNA荧光定量PCR的检测，更有利于HBV临床感染上的诊断、对治疗方案的选择及疗效评价。     

乙型肝炎患者3种血清标志物模式HBV DNA含量比较

目的比较乙型肝炎患者3种血清标志物模式HBV DNA含量。方法采用酶联免疫吸附试验(ELISA)检测乙型肝炎病毒(HBV)血清标志物,同份血清标本采用荧光定量PCR法检测HBV DNA。结果乙型肝炎患者血清学标志物模式均表现出一定的HBV DNA水平,尤以模式(HBsAg、HBeAg、抗-HBc阳性)HBV DNA含量最高,为107.16±1.79,且明显高于模式(HBsAg、抗-HBc阳性)(P0.01)和模式(HBsAg阳性、抗-HBe、抗-HBc阳性)(P0.05)。结论应用定量PCR法检测HBV DNA含量对反映HBV复制情况及其传染性强弱优于HBV血清学检测指标,对乙型肝炎患者决定是否接受治疗以及疗效监控也优于血清学标志物。     

实时荧光定量PCR测定TRAIL mRNA质粒标准品的构建

目的 构建pGEM-T-TRAIL重组质粒标准品为临床检测TRAIL mRNA水平奠定基础.方法 在TRAIL基因第二和第三外显子区设计特异性引物和荧光探针,提取血清HBV DNA阳性乙肝患者的外周血单个核细胞总RNA,逆转录合成cDNA,扩增目的 片断,纯化PCR产物与pGEM-T Easy载体连接,转化感受态菌DH - 5α,经蓝白斑筛选,PCR鉴定阳性克隆,并进一步测序分析.提取重组质粒测定DNA浓度后,10倍倍比稀释,建立TRAIL质粒标准品.结果 扩增目的 片段长度为110 bp,测序结果证实所构建的质粒序列与NCBI基因库TRAIL序列性一致.结论 成功构建了实时荧光定量PCR 测定TRAIL mRNA质粒标准品.     

慢性乙型肝炎患者外周血中共价闭合环状DNA的检测

目的 研究慢性乙型肝炎患者外周血中HBVcccDNA的存在状况.方法 采用荧光聚合酶链反应(PCR)法、PCR-微流芯片法对22例慢性乙型肝炎患者外周血血清、白细胞中HBVcccDNA进行平行检测,同时检测其血清HBV DNA含量、HBV M并进行相关性分析,以2例急性戊型肝炎、2例慢性丙型肝炎为对照.结果 22例慢性乙型肝炎患者外周血血清、白细胞中HBVcccDNA分别检出10例和7例,其中2例阳性PCR产物经基因测序得到验证;血清HBV DNA阳性者16例;HBVcccDNA阳性者血清HBV DNA均阳性;对照组上述指标均阴性.结论 慢性乙型肝炎患者外周血血清、白细胞中存在HBVcccDNA;外周血HBVcccDNA与血清HBV DNA存在一定关联.     

乙型肝炎病毒携带产妇进行乳汁H BV DN A检测对哺乳的指导价值

[目的]探讨乙型肝炎病毒携带产妇乳汁中乙型肝炎病毒DNA（HBV DNA）携带状况与母乳喂养安全性的关系,从而正确指导乙型病毒性肝炎产妇产后母乳喂养。[方法]采用酶联免疫吸附试验（ELISA）检测280例 HBV 携带产妇血清 HBV 免疫血清学标志物,将其分为乙型肝炎病毒 e抗原（HBeAg）阳性组和 HBeAg阴性组,同时采用荧光定量 PCR法检测血清及乳汁 HBV DNA含量。[结果]280例病人血清中 HBV DNA阳性172例（61.4%）,乳汁 HBV DNA阳性128例（45.7%）;血清 HBV DNA阳性组172例产妇乳汁中检测出 HBV DNA阳性124例（72.1%）,108例血清 HBV DNA阴性者中2例（1.8%）乳汁 HBV DNA阳性;HBeAg阳性组122例检测出血清 HBV DNA 阳性105例、乳汁 HBV DNA 阳性96例;HBeAg 阴性组158例检测出血清HBV DNA阳性67例、乳汁 HBV DNA阳性30例。[结论]在乙型肝炎病毒携带者母乳喂养问题上,应以 HBV DNA 检测为参考标准;乳汁 HBV DNA含量与血清 HBV DNA含量呈正相关,对血 HBV DNA阳性产妇,建议人工喂养,并母婴隔离;对血清HBV DNA阴性产妇可以哺乳,但应给予正确的哺乳指导。     

乙型肝炎病毒感染者HBeAg模式与病毒DNA水平相关性研究

目的 探讨乙型肝炎病毒(HBV)感染者HBeAg模式与HBV DNA水平之间的关系.方法 运用荧光定量PCR(FQ-PCR)检测120例乙型肝炎患者血清中的HBV DNA,同时运用ELISA方法进行HBV血清标志物的检测,并对结果进行相关性探讨.结果 HBeAg阳性组血清HBV DNA检出率为87.9%(58/66),...     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

Interprofessional approaches to creating safe,high quality health care

Three supplementary perspectives are presented arguing that interprofessional collaboration is both necessary and desirable. Nonetheless, there are often too many serious intra-professional barriers and obstacles to interprofessional collaboration to make it successful. Some of these barriers, it is argued and illustrated, are found in the multiple ways in which professional identity is tacitly acquired and embodied in the practitioners' habitual, everyday practice. The paper then explores ways in which reflection, especially Second order reflection, can help to elucidate and overcome these obstacles, as well as increasing professional adaptability and competence.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Prospects for the control of neglected tropical diseases by mass drug administration

The prospects for the control of neglected tropical diseases, including soil-transmitted helminthiasis, shistosomiasis, lymphatic filariasis, onchocerciasis and trachoma, through mass drug administration, are exemplified by the elimination of the trachoma as a public-health problem in Morocco. In spite of this and other striking successes, mass drug administration programs are faced with major challenges resulting from suboptimal coverage and lack of efficacy. At current suboptimal coverage rates, programs may need prolongation for an extended period, increasing costs and undermining sustainability. Community participation through health education and information appears to be crucial to improve coverage and to achieve sustainability. Implementation of complementary measures, such as vector control, improved hygiene and environmental sanitation, are important to further control transmission and to prevent re-emergence of the infection and, again, may only be achieved effectively through community-based initiatives. To reduce costs and to relieve pressure on the health system, combining neglected tropical disease programs in areas where diseases coexist and integration with existing control programs for malaria, tuberculosis and HIV/AIDS is advocated. The risk of developing drug resistance is of particular concern in view of the lack of alternative drugs, and reduced treatment efficacy due to emerging resistance is evident for the soil-transmitted helminths and onchocerciasis. Given the risk for the development of drug resistance and the need for a high degree of participation, close attention should be paid to the monitoring of the coverage and efficacy of the different program components.     

Bacterial meningitis: an update of new treatment options

The outcome of bacterial meningitis critically depends on the rapid initiation of bactericidal antibiotic therapy and adequate management of septic shock. In community-acquired meningitis, the choice of an optimum initial empirical antibiotic regimen depends on the regional resistance patterns. Pathogens resistant to antibacterials prevail in nosocomial bacterial meningitis. Dexamethasone is recommended as adjunctive therapy for community-acquired meningitis in developed countries. In comatose patients, aggressive measures to lower intracranial pressure <20 mmHg (in particular, external ventriculostomy, osmotherapy and temporary hyperventilation) were effective in a case–control study. Although many experimental approaches were protective in animal models, none of them has been proven effective in patients. Antibiotics, which are bactericidal but do not lyse bacteria, and inhibitors of matrix metalloproteinases or complement factor C5 appear the most promising therapeutic options. At present, vaccination is the most efficient method to reduce disease burden. Palmitoylethanolamide appears promising to enhance the resistance of the brain to infections.     

Prise en charge des entorses externes de cheville : étude clinique préliminaire sur l’efficacité du dispositif Myolux™

Ankle sprains are the most common injury of the musculoskeletal system and are associated with significant societal and economic impacts. It has been proven that classical therapeutic strategies may not be effective in preventing recurrent injuries: the recurrence rates reported in the literature can reach 73%. In order to provide an effective rehabilitation solution, a destabilizing orthosis was developed. This device is equipped with a mechanical articulator reproducing the subtalar mechanics and placed under the heel. In this paper, we present the main results of a preliminary clinical study conducted between 2004 and 2007. All subjects included in this study were treated with the abovementioned orthosis during 10 rehabilitation sessions of 30 minutes each. Data show a relatively low recurrence rate of 12% for the overall population. Moreover, it's of primary importance to note that this satisfactory ratio is largely reduced (3% of recurrence rate) for the 29 patients who performed one training session per month after the 10th initial rehabilitation sessions. Hence, the destabilizing orthosis appears to be an effective solution to prevent recurrent ankle sprains. However, joint protection requires long-term and regular training sessions. This result has motivated the development of a similar device allowing patients to perform training sessions at home. Finally, data obtained in this study are promising awaiting the final results of the comparative, multicentric and independent clinical trials currently managed by the Hospices Civils de Lyon.     

Imaging choices in occult hip fracture

Background: Hip fracture is a common injury, with an incidence rate of > 250,000 per year in the United States. Diagnosis is particularly important due to the high dependence on the integrity of the hip in the daily life of most people. Objectives: In this article we review the literature focused on hip fracture detection and discuss advantages and limitations of each major imaging modality. Discussion: Plain radiographs are usually sufficient for diagnosis as they are at least 90% sensitive for hip fracture. However, in the 3–4% of Emergency Department (ED) patients having hip X-ray studies who harbor an occult hip fracture, the Emergency Physician must choose among several methods, each with intrinsic limitations, for further evaluation. These methods include computed tomography, scintigraphy, and magnetic resonance imaging. Conclusion: We present an evidence-based algorithm for the evaluation of a patient suspected to have an occult hip fracture in the ED. Also outlined are future directions for research to distinguish more effective techniques for identifying occult hip fractures.     

Systematic Reviews Published in the April 2012 Issue of the Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD but monthly online. The April 2012 issue (second DVD for 2012) contains 5045 complete reviews, 2182 protocols for reviews in production, and 17,084 short summaries of systematic reviews published in the general medical literature. In addition, there are citations of 674,000 randomized controlled trials, and 15,400 cited papers in the Cochrane methodology register. The health technology assessment database contains just over 11,000 citations. One hundred and seventeen new reviews have been published in the last 3 months of which 12 have potential relevance for practitioners in pain and palliative medicine. The impact factor of the Cochrane Library stands at 6.186. Readers are encouraged to access the full report for any articles of interest as only a brief commentary is provided.     

Platelets,thrombosis, coagulation,and atherosclerosis

When I first got the invitation to join a medical delegation going to Moldova, I thought for a moment that our destination was the fictional country in the old Marx Brothers movie Duck Soup. On further checking, it turns out that entertaining place was called Freedonia. I now know that Moldova is indeed a real country, bordered on the west by Romania and on the other three sides by the Ukraine. It is a proud country, rich with traditions, and its people are warm, giving, eager to learn ways to improve their healthcare system, and deeply appreciative of our attempts to help them in the task.