2010-2015年湖北省乙型流感病毒流行和进化分析

目的 了解2010-2015年湖北省乙型流感病毒流行分布和基因进化情况。方法 通过测序和全球流感共享数据库（global initiative on sharing all influenza data,GISAID）获得2010-2015年湖北省乙型流感病毒血凝素（hemagglutinin, HA）和神经氨酸酶（neuramidinase, NA）氨基酸序列,分别对其进行系统进化树、氨基酸突变位点和三维建模分析,结合同期病毒分离株流行分布,分析重配株基因进化与病毒流行分布间的关系。结果 2010-2015年湖北省乙型流感病毒存在系内重配和系间重配,首例重配株出现后,该病毒都会形成较大流行。同期病毒在HA四个主要抗原表位上发现的突变位点有:N116K、N129S、A146T、K162R和N197S,在NA上发现了神经氨酸酶活性突变位点D197N,三维建模表明该位点并非直接与底物或抑制物接触。结论 2010-2015年湖北省乙型流感病毒的流行与该病毒的基因进化关系密切,监测该病毒抗原表位突变位点和耐药突变位点有助于深入分析其进化特性。     

Ⅰ类整合子在多重耐药铜绿假单胞菌中的分布与结构分析

目的 了解分离于镇江地区的铜绿假单胞菌对13种常用抗生素的耐药率;明确Ⅰ类整合子基因盒结构及其在耐药基因播散中的作用.方法 采用K-B纸片法检测71株铜绿假单胞菌的耐药率;煮沸法提取71株铜绿假单胞菌基因组DNA;PCR扩增Ⅰ类整合子基因,并通过测序分析其所携带耐药基因盒.结果 71株铜绿假单胞菌对临床常用的13种抗生素的耐药率在18.3%～77.5%不等;Ⅰ类整合子检出率为38%,包括aadB、aac(6')-Ⅱ、PSE-Ⅰ、dfrA17和aadA5 5种基因盒,其中最常见者为dfrA17和aadA5.Ⅰ类整合子阳性菌株对哌拉西林、哌拉西林/他唑巴坦、头孢曲松、头孢吡肟、头孢他啶、庆大霉素、阿米卡星、妥布霉素、左氧氟沙星、环丙沙星等10种抗生素的耐药率明显高于整合子阴性菌株.结论 不同铜绿假单胞菌临床株对13种常用抗生素的耐药率各不相同,整合子阳性菌株耐药率明显高于整合子阴性菌株,提示Ⅰ类整合子是铜绿假单胞菌多重耐药的重要因素.     

泌尿生殖道分离无乳链球菌药物敏感性分析

目的了解泌尿生殖道无乳链球菌感染及耐药状况,为临床用药提供依据。方法对临床送检的泌尿生殖道标本常规培养鉴定,并对分离出的无乳链球菌进行纸片扩散法药敏试验,对红霉素耐药、克林霉素敏感的菌株做D试验检测。结果144株无乳链球菌(尿77株,前列腺液36株,阴道分泌物31株)药敏结果显示,对万古霉素、利奈唑胺、青霉素和头孢曲松的耐药率最低,全部144株无乳链球菌中未发现耐药株;左氧氟沙星的耐药率较低,为16.2%;红霉素和克林霉素耐药率较高,分别为56.2%和53.5%。D试验阳性率为26.7%。结论从泌尿生殖道分离的无乳链球菌对青霉素、氨苄西林和头孢菌素类抗菌药物的敏感性较高,但对大环内酯类和克林霉素已有一定的耐药。     

中西医结合治疗耐药性肺结核病的临床观察

目的探讨中西医结合治疗耐药性肺结核病的临床疗效。方法选择门诊复治耐药肺结核病患者80例,分成治疗组和对照组。两组患者在给予相同抗结核药物治疗的基础上,治疗组40例,加服中药养阴润肺汤,对照组40例只给予口服抗结核药物治疗,疗程均为1个月。健康对照组40人外周血做对照。结果治疗组痰涂片和痰结核分枝杆菌培养的阴转、病灶吸收好转、血沉恢复正常与对照组比较差异有统计学意义（P〈0.05）;外周血中T淋巴细胞亚群变化治疗前、后比较差异有统计学意义（P〈0.05）,对照组治疗前后差异无统计学意义（P〉0.05）。结论中西医联合用药可以更好地改善耐药肺结核病患者免疫状况,促进临床症状的改善。     

Analysis of hepatitis B virus drug-resistant mutant haplotypes by ultra-deep pyrosequencing

Direct sequencing and reverse hybridization are currently the main methods for detecting drug-resistance mutations of hepatitis B virus (HBV). However, these methods do not enable haplotype analysis so they cannot be used to determine whether the mutations are co-located on the same viral genome. This limits the accurate identification of viral mutants that are resistant to drugs with a high genetic barrier. In our current study, ultra-deep pyrosequencing (UDPS) was used to detect HBV drug-resistance mutations in 25 entecavir-treated and five treatment-naive patients. Of the 25 entecavir-treated patients, 18 had experienced virological breakthrough and two exhibited reduced susceptibility to entecavir. The results obtained by UDPS were compared with those of direct sequencing, and the haplotypes of the drug-resistant HBV mutants were analysed. The average number of reads per patient covering the region in which drug-resistance mutations are located was 1735 (range 451-4526). UDPS detected additional drug-resistance mutations not detected by direct sequencing in 19 patients (mutation frequency range 1.1-23.8%). Entecavir-resistance mutations were found to be co-located on the same viral genome in all 20 patients displaying virological breakthrough or reduced susceptibility to entecavir. In conclusion, UDPS was not only sensitive and accurate in identifying drug-resistance mutations of HBV but also enabled haplotype analysis of the mutants. This method may offer significant advantages in explaining and predicting the responses of patients with HBV to antiviral therapy.     

Conversion from immediate-release to extended-release lamotrigine improves seizure control

BackgroundImmediate release lamotrigine (LTG-IR) dosing can be limited by peak toxicity. It is thought that peak levels are responsible for some adverse effects such as dizziness, blurred vision, double vision and unsteadiness. At the same time, trough levels may be associated with reduced seizure threshold. The use of extended release lamotrigine (LTG-XR) to replace LTG-IR will be associated with less fluctuation in drug levels-lower peak levels may reduce adverse effects and higher trough levels may improve seizure control. This hypothesis was tested by analyzing seizure control and adverse effects before and after conversion from LTG-IR to LTG-XR in patients who underwent such conversion in 2009–2011.MethodsWe searched our patient database to identify patients converted from LTG-IR to LTG-XR for persistent seizures or adverse effects from August 2009 until December 31, 2011. We included only patients who took LTG-IR and LTG-XR for at least 6 months each. We excluded patients with nonepileptic seizures, progressive cause of epilepsy, or not keeping a seizure record. We collected the following parameters: age at conversion, LTG-IR dose and dosing schedule, duration on that dose, LTG-XR dose and dosing schedule, LTG serum level before and after conversion, duration of LTG-XR treatment, seizure frequency before and after conversion, and change in adverse experience profile. We also recorded baseline AEDs and any AED change during the course of the analysis.ResultsFifty five patients (26 female) satisfied the inclusion/exclusion criteria. Their mean age was 45 years (range 23 to 86). Ten were on LTG-IR monotherapy, 24 took LTG-IR plus one other AED, most commonly levetiracetam, and the remaining 21 took LTG-IR plus at least 2 other AEDs. The mean LTG-IR dose was 544 mg/day (range 150–1100 mg/day). The mean LTG-IR serum level was 11.6 (available in 53 patients-range 4.6–21 mcg/ml). Twenty six patients were converted to the same dose and one patient took a mixture of LTG-XR and LTG-IR at the same total daily dose, while 21 had their dose slightly increased and 7 had their dose slightly decreased due to adverse effects. The mean serum level after conversion was 11.8 (available in 49 patients-range 2.6–21.2 mcg/ml). As a result of the conversion, 26 patients (47%) experienced >50% reduction in seizure frequency. There was a 46% median reduction in seizure frequency overall. Seven patients reported improvement in adverse effects.ConclusionA conversion from LTG-IR to LTG-XR can help improve seizure control in some individuals with drug-resistant epilepsy, in addition to improving tolerability. While it is indicated in individuals experiencing peak adverse effects, it should also be considered in patients who have received incomplete seizure control from LTG-IR.     

A single chimpanzee-human neutralizing monoclonal antibody provides post-exposure protection against type 1 and type 2 polioviruses

BackgroundDevelopment of anti-poliovirus therapies to complement vaccination is an urgent priority. A number of antiviral drugs are in development. Recently we have developed human monoclonal antibodies that could be used for treatment of chronically infected individuals and emergency response to potential reappearance of polioviruses after eradication.ObjectiveThe aim of this study was to characterize neutralizing activity of anti-poliovirus monoclonal antibody A12 against wild type, vaccine-derived, and drug-resistant poliovirus strains, evaluate in vivo pre- and post-exposure protective properties of the antibody against polioviruses of serotypes 1 and 2, and to determine whether it interferes with response to immunization with poliovirus vaccine.Study designImmunogenicity studies were performed in CD1 mice. Poliovirus neutralizing titers were determined in poliovirus microneutralization assay. Poliovirus immunization-challenge experiments were performed in poliovirus-susceptible TgPVR21 mice.ResultsWe show that monoclonal antibody A12 effectively neutralizes in vitro a broad range of type 1 and type 2 wild and vaccine-derived polioviruses, provides effective pre- and post-exposure protection of TgPVR21 mice from challenge with a lethal dose of poliovirus. Treatment of animals with the antibody concurrent with IPV immunization does not prevent immune response to the vaccine.ConclusionsAnti-poliovirus antibody A12 effectively neutralizes a range of wild and VDPV strains and protectstransgenic mice susceptible to poliovirus against lethal challenge upon pre- and post-exposure administration. This suggests that the antibodies could be used in combination with drugs and/or vaccine to improve their efficacy and prevent emergence of resistant variants, and provides a justification for initiating their clinical evaluation.     

2012-2017年某院耐碳青霉烯肺炎克雷伯菌变迁及耐药性

目的 分析医院耐碳青霉烯肺炎克雷伯菌(CRKP)的流行趋势、分布特点及耐药性,为CRKP的防治提供临床依据。方法 调查某院2012-2017年住院患者抗菌药物使用情况及CRKP分离率和耐药数据,采用χ2检验、Spearman相关性检验等进行分析。结果 6年共分离肺炎克雷伯菌1911株,其中280株为CRKP。CRKP检出率由2012年的0.4%上升至2017年的29.5%;重症监护病房(ICU)CRKP的分离率明显高于普通病房(P<0.05);CRKP检出与碳青霉烯类、第三代头孢菌素和青霉素酶抑制剂复方制剂的使用强度呈明显的正相关(P<0.05)。CRKP株对大多数抗菌药物的耐药率超过90%。结论 该院CRKP分离率较高,与入住ICU及广谱β-内酰胺类、碳青霉烯类的用量有关,呈现多重耐药性。应加强抗菌药物管理,强化消毒、隔离等医院感染控制措施,以减少耐药株的播散和流行。     

耐碳青霉烯类肠杆菌科细菌的分布及耐药性分析

目的：分析耐碳青霉烯类肠杆菌科细菌的分布情况及耐药性。方法：收集丽水市中心医院2010年12月～2013年6月临床分离的耐药菌株,采用法国梅里埃Vitek-2 compact细菌鉴定仪鉴定细菌,药物敏感性试验采用纸片扩散法（K-B法）。结果：共收集耐药菌株83株,其中肺炎克雷伯菌占68.67%,产气肠杆菌和阴沟肠杆菌各6.02%,大肠埃希菌2.41%。标本来源最常见为痰液（80.72%）,其次尿液（8.43%）和分泌物（4.82%）,科室分布最多者为重症监护病房（39.76%）,其次为神经外科（36.51%）。74例患者在检测出耐药菌株前1个月内使用过至少一种抗菌药。有69.88%和31.33%的患者使用过哌拉西林/他唑巴坦和头孢哌酮/舒巴坦,15.66%和10.84%使用过亚胺培南/西司他丁和美罗培南。耐药菌株对多黏菌素B高度敏感,对米诺环素和阿米卡星的耐药率为13.25%和28.92%,对其他抗菌药的耐药率多介于70%～100%。结论：耐碳青霉烯类肠杆菌科细菌在医院重症监护病房和神经外科相对集中,对多数临床常用抗菌药呈高度耐药,给临床治疗带来严重的挑战。医院应做好院感监测,合理使用抗菌药,从而有效预防和控制耐药菌株的产生和传播。     

儿童肺炎支原体的耐药性与临床用药相关性研究

目的 探讨阿奇霉素序贯疗法治疗儿童肺炎支原体（MP）感染的有效性和安全性,并检验不规律使用阿奇霉素与MP耐药性是否相关.方法 以MP抗体滴度1∶320为诊断MP感染标准,收集2011年3月至2013年2月于广州医科大学附属广东省妇女儿童医院住院治疗的792例5～13岁合并发热或（和）咳嗽的MP感染患儿中,MP-IgM呈阳性的431例患儿为研究对象.按照其1年内大环内酯类抗菌药物应用和MP感染次数分别纳入实验组（n=217,半年内无MP感染和大环内酯类抗菌药物应用史）;对照组（n=214,1年内反复MP感染且不规律应用大环内酯类抗菌药物≥2次）.患儿入院当天于不同时间进行2次MP培养,对培养结果MP呈阳性者提取DNA进行23S rRNA V PCR产物合成与23S rRNA V区基因突变位点检测,并进行MP呈阳性者的9种抗菌药物的药敏性试验.对两组MP株的耐药性等进行比较.对大环内酯类抗菌药物敏感和23S rRNA V区无突变患儿进行阿奇霉素序贯疗法.于治疗2周及4周末再行MP培养,对阳性者分析23S rRNA V区基因位点突变情况.对实验组阿奇霉素敏感且23S rRNA V区测序无基因位点无突变的患儿进行阿奇霉序贯治疗,分别于治疗2周及4周时判断阿奇霉素序贯疗法治疗疗效.对于对照组214例反复MP感染患儿不规律应用大环内酯类抗菌药物频次与耐药性关系进行统计学比较.对两组患儿的肺外并发病及住院时间分别进行统计学比较（本研究遵循的程序符合广州医科大学附属广东省妇女儿童医院人体试验委员会制定的伦理学标准,得到该委员会批准,分组征得受试对象监护人的知情同意,并与其签署临床研究知情同意书）.两组患儿年龄及性别等一般临床资料比较,差异无统计学意义（P〉0.05）.结果 本组患儿的MP感染率为54.4%（431/792）.对本组药敏实验结果MP-IgM呈阳性患儿咽拭子进行快速MP培养的阳性率为26.6%（115/431）.实验组与对照组MP株耐药率比较,差异有统计学意义（χ2=4.651,P=0.041）.对照组214例患儿1年内不规律大环内酯类药物应用频次与耐药率比较,不规律用药次数越多,耐药率越高,差异有统计学意义（χ2/χ2趋势值=22.056,21.932;P〈0.05）.实验组14例患儿的药敏实验结果显示阿奇霉素敏感且23S rRNA V区测序无基因位点无突变,对其进行阿奇霉序贯治疗2周后,咽拭子培养结果显示MP呈阳性为4例,根治率为71.42%（10/14）.对此4例进行药敏试验结果提示对阿奇霉素仍敏感,并对其进行23S rRNA V区测序发现无基因位点突变,继续阿奇霉素序贯治疗,至4周末时,实验组14例患儿的根治率为92.85%（13/14）.实验组14例MP药物敏感株与对照组37例MP耐药株患儿的肺外并发症发生率比较,实验组显著低于对照组,且差异有统计学意义（χ2=4.443,P〈0.05）.实验组患儿的住院时间显著短于对照组,且差异有统计学意义（χ2=7.305,P〈0.05）.结论 阿奇霉素序贯疗法治疗儿童MP感染安全有效,并不会诱导出耐药株,而不规律应用阿奇霉素可诱导耐药株,且耐药率与不规律应用的次数呈正相关.