High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA.

The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.     

EFFICACY OF RAMIPRIL VERSUS ENALAPRIL IN PATIENTS WITH MILD TO MODERATE ESSENTIAL HYPERTENSION

SUMMARY This double-blind, randomised, cross-over study investigated the antihypertensive efficacy of ramipril and enalapril was completed by 30 patients with mild-to-moderate essential hypertension. After a four-week placebo run-in phase, the patients received either 2.5mg ramipril or 10mg enalapril once daily for four weeks. The dosages were increased to 5mg ramipril and 20mg enalapril for a further four weeks. After a placebo washout phase of four weeks, the patients were crossed over to the alternative treatment. The decrease in average 24-hour ambulatory diastolic blood pressure from week 0 to week 8 was 1.6mmHg greater with ramipril than enalapril (90% confidence interval 0.6-2.7mmHg). The corresponding reduction in for systolic blood pressure was also greater with ramipril than enalapril by 2.4mmHg (90% confidence interval: 0.5-4.2mmHg). For the difference in the drop of 24-hour ambulatory diastolic blood pressure between ramipril and enalapril the lower level of the 90% confidence interval (CI) is above the clinically relevant difference of -3mmHg. This is an indication that ramipril (2.5 and 5mg dose) is at least as effective as enalapril (10 and 20mg dose) in decreasing blood pressure in patients with mild-to-moderate essential hypertension. The duration of adequate antihypertensive effect was relatively long for both ramipril and enalapril; however, ramipril tended to have a more prolonged antihypertensive effect. Ramipril had a higher diastolic and systolic trough/peak ratio than enalapril, resulting in a more uniform antihypertensive effect over the 24-hour treatment period. Both ramipril and enalapril were well tolerated and the two treatment groups had similar safety profiles.     

Pre-B cells and other possible precursor lymphoid cell lines derived from patients with X-linked agammaglobulinemia

A group of unique Epstein-Barr virus-containing cell lines was derived from the bone marrow of three patients with X-linked agammaglobulinemia. Efforts to obtain cell lines from the peripheral blood of these patients were uniformly unsuccessful. Immunofluorescence analyses as well as biosynthetic studies with [(35)S]methionine indicated unusual patterns of Ig synthesis in many of these bone marrow derived lines. Seven of the lines were of particular interest in that two produced no Ig of any type; two others showed no Ig by fluorescence but small amounts by [(35)S]methionine labeling; one expressed only cytoplasmic μ chains without any evidence of light chain synthesis, and two produced primarily μ chains with only slight amounts of light chains. One of the lines without membrane or cytoplasmic Ig studied in detail grew like a typical lymphoid line and was carried in intermittent culture over a period of 2 yr without Ig expression. One line grew quite differently and resembled the round cell type described previously, which has been obtained from a variety of sources. The cell line with cytoplasmic μ chains and no light-chain expression had the characteristic properties of pre-B cells. Three normal type Ig-producing cell lines also were obtained from the patients. The accumulated evidence obtained in the present study indicates that these unusual cell lines represent normal precursor cells of the B-cell lineage; these grew out in these cases because of the virtual absence of mature B cells that ordinarily overgrow the culture system. However, the possibility that in certain instances they reflect abnormal Ig synthesis characteristic of the disease has not been ruled out.     

The win ratio method in heart failure trials: lessons learnt from EMPULSE

Aims

The EMPULSE trial evaluated the clinical benefit of empagliflozin versus placebo using the stratified win ratio approach in 530 patients with acute heart failure (HF) after initial stabilization. We aim to elucidate how this method works and what it means, thereby giving guidance for use of the win ratio in future trials.

Methods and results

The primary trial outcome is a hierarchical composite of death, number of HF events, time to first HF event, or a ≥5-point difference in Kansas City Cardiomyopathy Questionnaire (KCCQ) total symptom score change at 90 days. In an overall (unstratified) analysis we show how comparison of all 265 x 265 patients pairs contribute to ‘wins’ for empagliflozin and placebo at all four levels of the hierarchy, leading to an unstratified win ratio of 1.38 (95% confidence interval [CI] 1.11–1.71; p = 0.0036). How such a win ratio should (and should not) be interpreted is then described. The more complex primary analysis using a stratified win ratio is then presented in detail leading to a very similar overall result. Win ratios for de novo acute HF and decompensated chronic HF patients were 1.29 and 1.39, respectively, their weighted combination yielding an overall stratified win ratio of 1.36 (95% CI 1.09–1.68) (p = 0.0054). Alternative ways of including HF events and KCCQ scores in the clinical hierarchy are presented, leading to recommendations for their use in future trials. Specifically, inclusion of both number of HF events and time-to-first HF event appears an unnecessary complication. Also, the use of a 5-point margin for KCCQ score paired comparisons is not statistically necessary.

Conclusions

The EMPULSE trial findings illustrate how deaths, clinical events and patient-reported outcomes can be integrated into a win ratio analysis strategy that yields clinically meaningful findings of patient benefit. This has implications for future trial designs that recognize the clinical priorities of patient evaluation and the need for efficient progress towards approval of new treatments.     

Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.     

理科大学生网络成瘾与人格特质的关系

目的:分析理科大学生网络成瘾与其人格特质的关系。方法:于2005-09/11在上海某高校以班级为单位,对4个理科专业的220名学生进行问卷调查,4个专业分别是数学教育、计算机教育、物理师范和应用心理学。采用大学生因特网成瘾量表、交往焦虑量表、UCLA孤独量表和YG性格测验等4份问卷进行调查。大学生上网情况调查表共有52个题目,5级计分,成瘾诊断标准有耐受性、脱瘾综合症、计划性、控制性、行为特征、危害性、主观认识和行为等7个维度。按1,2,3,4,5记分,即每题填什么数记什么分,然后将各题得分相加得到维度分。判断标准:每题得1～3分转化为0分,4分转化为1分,5分转化为2分。测验每一部分导出分相加。若测验7部分中有3部分以上得分超过4分,则可基本诊断该大学生为因特网成瘾障碍患者。交往焦虑量表包含15个自陈条目,5级计分,总分从15(社交焦虑程度最低)到75(社交焦虑程度最高)。UCLA孤独量表(第3版)包含20个自陈条目,4级计分,总分从20(孤独程度最低)到80(孤独程度最高)。需要补充说明的是,交往焦虑量表是测量独立于行为的社交焦虑,UCLA孤独量表也主要是特质量表,因此这两个量表所测量的都是焦虑和孤独的人格特质而不是状态。YG性格测验问卷包含12个分量表,每个分量表有10个题目,测量一种特质。结果:共发出220份问卷,收回有效问卷211份。①成瘾者和非成瘾者在焦虑和孤独量表上的得分差异没有显著性意义。②成瘾者和非成瘾者在YG性格测验中的抑郁性、循环性、神经质、非合作性和攻击性等特质得分上,差异十分显著[(9.81±4.97),(5.95±5.10)分;(10.81±4.56),(6.78±4.46)分;(9.63±4.72),(6.51±4.67)分;(11.15±4.19),(7.28±4.43)分;(12.41±4.05),(8.69±3.69)分,均P<0.001],自卑感、主观性和细致性等特质得分上差异显著[(9.15±4.51),(6.83±4.49)分;(10.04±3.50),(7.63±4.09)分;(12.67±3.45),(10.26±4.23)分,P<0.01或P<0.05],在一般活动性、思维外向性、支配性和社会外向性等特质得分上两者没有差异。结论:理科大学生的部分人格特质和网络成瘾密切相关。     

缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化

目的:制备大鼠在体缺血再灌注模型,观察缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化。方法:实验于2005-03/2006-10在解放军沈阳军区总医院医学实验动物中心和全军心血管研究所实验室完成。实验分组:选用健康雌性SD大鼠36只,根据预适应程序分为第1,2,3次缺血,第1,2,3次再灌注,每一时间点6只大鼠。实验过程:用手术套管法造成左冠状动脉主干缺血及再灌注。所有实验动物在实验程序结束后,取出心脏迅速置液氮保存备用。实验评估:用放射免疫法测环磷酸腺苷水平,生化法测环磷酸腺苷依赖蛋白激酶活性变化。结果:36只大鼠均进入结果分析。①环磷酸腺苷含量:第1次再灌注组低于第1次缺血组[(0.325±0.015),(0.395±0.024)pmol/g,t=6.06,P<0.001],第2次再灌注组低于第2次缺血组[(0.523±0.017),(0.708±0.067)pmol/g,t=6.56,P<0.001],第3次再灌注组低于第3次缺血组[(0.567±0.031),(0.712±0.038)pmol/g,t=7.24,P<0.001]。②环磷酸腺苷依赖蛋白激酶活性:第1次再灌注组低于第1次缺血组[(10.115±1.000),(16.351±0.849)pkat/g,t=11.12,P<0.001],第2次再灌注组低于第2次缺血组[(11.877±2.213),(14.869±0.619)pkat/g,t=3.31,P<0.01],第3次再灌注组低于第3次缺血组[(11.745±0.987),(14.766±0.329)pkat/g,t=7.09,P<0.001]。③缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性随缺血及再灌注呈周期性波动。在5min缺血预处理时表现为明显增高,而在间隔的再灌注程序中恰呈相反改变,有明显下降的趋势。结论:环磷酸腺苷及环磷酸腺苷依赖蛋白激酶的周期性波动变化可能是激发心肌缺血预处理的机制之一,环磷酸腺苷可能在预处理保护作用中起一些作用。     

股方肌肌骨瓣植入治疗成人股骨头缺血性坏死

目的:目前成人股骨头缺血性坏死的手术方法较多,但远期疗效大多不肯定。股方肌肌骨瓣植入术可以治疗成人股骨头缺血性坏死,但需验证其近远期疗效。方法:选择2001-01/2007-01铜川市矿务局中心医院骨科收治的股方肌肌骨瓣植入治疗股骨头坏死患者15例(18髋),均知情同意。术中暴露股方肌及其在股骨近端的附着点,于附着点处凿取骨瓣,骨瓣为4cm×1.5cm×1.0cm的长方形,将骨瓣插入股骨头内,远端用可吸收骨钉固定。术后3,6,12,24个月门诊复查拍患髋正位和蛙式位X射线片,根据临床查体和X射线片表现将手术效果分为优、良、差3级。结果:全部患者均获随访,随访时间4～36个月。近期疗效满意,出院时疼痛症状均缓解,未见手术相关并发症。中远期随访结果优10髋,良6髋,差2髋,优良率88.9%。结论:股方肌肌骨瓣植入术治疗成人股骨头缺血性坏死近远期疗效确切,手术操作相对简单。     

Isolation and characterisation of Ralstonia solanacearum strains from Solanaceae crops in Ethiopia

Eighty one isolates of Ralstonia solanacearum -like bacteria on triphenyl tetrazolium chloride (TTC) medium were collected from different Solanaceae crops (i.e. potato, tomato and pepper plants and potato tubers) at various sites in Ethiopia. Of these, 62 strains were identified as R. solanacearum based on their cultural characteristics on TTC medium, tomato pathogenicity bioassay, carbon source utilisation patterns and a specific PCR-based assay. By Hayward's classification method, based on carbon source utilisation, 19 of the 62 R. solanacearum strains were identified as biovar I and 43 strains were identified as biovar II. The biovar I strains exhibited a high growth rate at high temperatures (37 degrees C). Whereas the growth rate of biovar II strains was greatest at lower temperatures (22 degrees C). Biovar I strains had broader host range than biovar II strains, which were limited to potato, tomato, and eggplant. To our knowledge, this is the first report of R. solanacearum biovar I in Ethiopia. The existence of biovar I strains in Ethiopia raises concerns because they have a broader host range than biovar II strains.     

Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), micro -capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.