肺炎嗜衣原体诊断候选抗原的研究进展

肺炎嗜衣原体（Cpn）是二十世纪八十年代新发现的一种重要的呼吸系统感染的病原体,它不仅可以引起非典型肺炎,而且还有可能通过直接或间接的机制参与动脉粥样硬化的发生与发展,因而受到了人们越来越多的关注。近年来,Cpn感染的血清学诊断也成为人们关注的焦点之一,目前常用于诊断Cpn感染的抗原有全菌抗原、脂多糖和主要外膜蛋白等属特异性抗原。因此本文就Cpn感染诊断候选抗原的研究进展作一综述。     

肺炎嗜衣原体蛋白酶样活性因子重组蛋白的血清学诊断价值初探

目的克隆、表达肺炎嗜衣原体（Cpn）蛋白酶样活性因子（CPAF）的免疫优势区基因（CPAFm），初步评价其在血清学诊断中的应用价值。方法构建pGEX6p-2／CPAFm重组质粒，诱导表达重组蛋白，十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定结果；将纯化蛋白免疫新西兰兔，间接酶联免疫吸附试验（ELISA）检测其免疫原性；与人抗Cpn抗血清反应分析其免疫反应性。间接ELISA法检测Cpn IgG和沙眼衣原体（Ct）阳性血清。结果高效表达和纯化出一相对分子质量约为51.3×10^3的重组蛋白，蛋白质印迹法（Western blot）证明其能与人抗Cpn IgC抗体发生反应；在被免疫的新西兰兔体内，特异性IgG抗体的滴度为1：16000以上，间接ELISA法检测40例Cpn IgG参考血清，阴性和阳性结果的符合率均为100．0%（20／20）；与微量免疫荧光法（MIF）对照，检测300例临床血清标本中的IgG抗体，符合率为98．3％；检测Ct阳性血清未见交叉反应。结论表达的Cpn CPAF免疫优势区重组蛋白具有良好的免疫活性，在Cpn的血清学诊断中具有较高的应用价值。     

自制肺炎衣原体抗原的临床应用

目的:评估自制肺炎衣原体抗原(Chlamydia pneumoniae-antigen,Cpn-Ag)用于肺炎衣原体(Chlamydia pneumoniae,Cpn)感染的诊断价值。方法:采用Dot-ELISA法观察自制Cpn-Ag与进口Cpn-单克隆抗体(McAb)的结合反应。采用微量免疫荧光法,用自制Cpn-Ag和进口Cpn-Ag同时检测268例血浆标本的Cpn-IgA、IgG和IgM。结果:Dot-ELISA结果显示,自制Cpn-Ag可与McAb发生特异性结合反应。自制Cpn-Ag与进口Cpn-Ag检测血浆中Cpn-IgA、IgG、IgM抗体滴度的一致率均达90%以上;以进口Cpn-Ag检测为标准,自制Cpn-Ag检测灵敏度、特异度均在90%以上;阳性似然比均>10,阴性似然比均<0.1。配对卡方检验显示,自制Cpn-Ag与进口Cpn-Ag检测血浆Cpn-IgA、IgG、IgM的结果比较,差异无统计学意义(P>0.05);一致性检验显示,Kappa值>0.75,两者具有高度一致性。结论:采用自制Cpn-Ag检测血浆Cpn-IgA、IgG、IgM,具有较高的灵敏度和特异度,与采用进口Cpn-Ag的检测结果具有高度一致性。     

肺炎嗜衣原体感染的诊断研究现状

肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)是于1989年确定的一种严格真核细胞内寄生的原核细胞型微生物,常引起呼吸道疾病的暴发流行[1]。Cpn感染遍及全球,据统计,包括美国、日本、匈牙利等在内的许多国家,人群Cpn抗体阳性者超过全国人口的一半以上,且Cpn感染因时间、地区、     

四种国产大环内酯类抗菌药物体外抗沙眼衣原体和肺炎衣原体敏感性研究

目的 研究国产必特螺旋霉素、乙酰螺旋霉素、红霉素和阿奇霉素等4种大环内酯类药物对于沙眼衣原体和肺炎衣原体体外药物敏感性试验,评估其抗衣原体作用,以指导临床用药.方法 细胞培养和免疫荧光包涵体染色技术测定4种国产大环内酯类抗菌药物对于沙眼衣原体和肺炎衣原体体外MIC.结果 对于沙眼血清型B,必特螺旋霉素、红霉素和阿奇霉素体外MIC为0.5μg/ml,乙酰螺旋霉素为4μg/ml.对于沙眼血清型D,必特螺旋霉素与阿奇霉素体外MIC均为0.25μg/ml,红霉素0.5μg/ml,乙酰螺旋霉素2μg/ml.对于肺炎衣原体,红霉素体外MIC≤0.016μg/ml,阿奇霉素和必特螺旋霉素均为0.032μg/ml,乙酰螺旋霉素0.5μg/ml.结论 国产必特螺旋霉素、红霉素和阿奇霉素体外抗沙眼衣原体(血清型B和D)和肺炎衣原体作用可靠,但乙酰螺旋霉素则较差.     

三种结核分枝杆菌重组蛋白抗原血清学诊断价值的研究

目的 研究重组ESTA6(rESTA6)、CFPl0(rCFP10)、CFP1O-ESTA6(rCFP10-ESTA6)三种结核分枝杆菌重组抗原在结核病血清学诊断中的应用价值.方法 检测230例肺结核、70例非结核疾病及200例健康血清标本中抗结核抗体,应用SPSS 18.0对检测结果进行方差分析.结果 方差分析表明,rCFP10、rESAT6、rCFP10-ESAT6三种抗原对肺结核、非结核疾病及健康人血清标本的检测结果有统计学意义(P<0.05),三种抗原检测肺结核病患者血清标本的敏感性分别为31.3%、25.2%、34.8%,特异性分别为94.8%、97.8%、95.6%.rCFP10和rESAT6联合检测敏感性及特异性分别为40.O%和93.O%;rCFPl0和rESAT6联合检测的92例阳性标本中,有23例rCFP10-ESAT6检测呈阴性;rCFP10-ESAT6检测的80例阳性标本中,有26例rCFP10和rESAT6联合检测呈阴性.结论 rCFP10-ESTA6的结构与rCFP10和rESTA6的单体的结构可能不完全相同,rCFP10-ESTA6抗原不能完全代替rESTA6、rCFP10两种抗原进行结核病的血清学实验室诊断.     

肺炎嗜衣原体Cpn 0147重组蛋白的免疫学活性研究

目的 研究Cpn 0147重组蛋白的免疫学活性及其应用于肺炎嗜衣原体(Cpn) 感染临床诊断中的价值.方法 采用GST琼脂糖凝胶纯化目的 蛋白,将Cpn 0147重组蛋白免疫新西兰兔,分别应用Western blot、ELISA法检测其免疫反应性及免疫原性,同时通过检测临床标本以评估其诊断价值.结果 成功表达并纯化了相对分子质量约41×103的重组蛋白GST-Cpn 0147;Western blot和ELISA结果显示,该重组蛋白具有良好的免疫反应性,动物实验结果表明该重组蛋白具有较好的免疫原性,ELISA结果显示该重组蛋白免疫新西兰兔血清抗体效价为1∶12 800.结论 Cpn 0147重组蛋白具有较好免疫学活性和特异性,可为Cpn感染的确诊、预防研究奠定基础.     

新生儿沙眼衣原体肺炎30例临床分析

目的：探讨沙眼衣原体（CT）所致新生儿肺炎的流行病学情况和临床特点。方法：收集150例肺炎新生儿的鼻咽分泌物,用聚合酶链反应方法检测CT。结果：CT感染阳性率为20%（30/150）,经阴道分娩儿CT感染率（29%,26/91）明显高于剖宫产儿（7%,4/59）（P〈0.01）,农村CT感染率（29%,17/59）高于城市（14%,13/91）（P〈0.05）。临床表现以咳嗽、气促、肺部罗音为主。结论：CT是新生儿肺炎的常见病原体,CT感染与分娩方式有关。     

患儿肺炎支原体、衣原体诊断价值

肺炎支原体（MP）和肺炎衣原体（CP）是引起呼吸系统感染常见的病原体,易引起人类多种疾病,在临床上越来越受到重视,因此早期检测患儿MP和CP血清中的IgM抗体可为临床诊断提供参考。     

重组结核分枝杆菌PPE65蛋白的制备及其在肺结核诊断中的价值

目的 评价重组PPE65蛋白IgG抗体用于检测肺结核患者的价值.方法 将编码结核分枝杆菌PPE65蛋白的基因克隆到PET-28a载体,并在大肠埃希菌中表达,镍亲和层析和离子层析法纯化重组PPE65蛋白,透析复性和Lowry法测蛋白浓度.采用ELISA法检测144例肺结核患者、144名健康者、56例非结核肺部疾病患者血清中抗重组PPE65蛋白和重组PstS1蛋白的IgG抗体水平.用ELISA检测144例肺结核患者和97名PPD皮试阴性健康人血清IgG结果绘制ROC曲线确定临界值.分析评价重组PPE65蛋白及其与重组PstS1蛋白联合检测肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性.结果 结核分枝杆菌PPE65蛋白在大肠埃希菌中获得表达,层析纯化获得纯度为95%的重组PPE65蛋白,复性后蛋白浓度为0.5 mg/ml.重组PPE65蛋白IgG抗体的ELISA检测临界值为0.64.重组PPE65蛋白诊断肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性分别为34.7%(50/144)、93.5%(187/200)、79.4%(50/63)、66.5%(187/281)、68.9%(237/344);重组PPE65蛋白和重组PstS1蛋白联合诊断肺结核的敏感度、特异度、阳性预测值、阴性预测值、准确性分别为59.0%(85/144)、91.0%(182/200)、82.5%(85/103)、75.5%(182/241)、77.6%(267/344).结论 结核分枝杆菌PPE65蛋白可作为结核患者血清检测的蛋白抗原之一.PPE65蛋白和重组PstS1蛋白IgG抗体联合检测用于结核病诊断可提高敏感度.

Abstract：

Objective To evaluate the potential value of IgG antibodies against recombinant PPE65 protein (rPPE65) of Mycobacterium tuberculosis in serodiagnosis of tuberculosis.Methods The gene encoding PPE65 protein of M.tuberculosis was cloned into the PET-28a vector and then expressed in Escherichia coli.The rPPE65 was purified with Ni-NTA affinity and ion exchange chromatography.After dialysis renaturation, the concentration of rPPE65 was determined using Lowry assay.ELISA was used to detect the levels of specific IgG against rPPE65 and recombinant PstS1 protein (rPstS1) in sera from 144 patients with pulmonary tuberculosis (PTB patients), 144 health controls, and 56 patients with non-tuberculosis pulmonary diseases.ROC curves were used to determine cut-off values with the results of IgG antibodies against rPPE65 and rPstS1 for 144 PTB patients and 97 controls with negative PPD skin test.The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of rPPE65 and the combination of rPPE65 and rPstS1 were counted.Results The PPE65 protein of M.tuberculosis was successfully expressed in E.coli. The purity and concentration of rPPE65 were 95% and 0.5 mg/ml, respectively.ROC analysis showed that the cut-off of ELISA using rPPE65 was 0.64.The sensitivity, specificity, PPV, NPV, and accuracy of rPPE65 were 34.7%(50/144), 93.5%(187/200), 79.4%(50/63), 66.5%(187/287), and 68.9%(237/344), respectively.The sensitivity, specificity, PPV, NPV, and accuracy of the combination of rPPE65 and rPstS1 were 59.0%, 91.0%, 82.5%, 75.5%, 77.6%, respectively.Conclusions The rPPE65 of M.tuberculosis appears to be a candidate antigen for serodiagnosis of tuberculosis.Detection of IgG antibodies against the combination of rPPE65 and rPstS1 can increase the sensitivity of serological test for tuberculosis.     

Comparative study of three different mycobacterial antigens with a novel lipopolysaccharide antigen for the serodiagnosis of tuberculosis

Demonstration of Mycobacterium tuberculosis in a smear or culture is the most reliable method for diagnosing tuberculosis (TB). In the last 10 years, several enzyme-linked immunosorbent assays (ELISAs) based on mycobacterial antigens (such as antigen 60, 38 kDa antigen, and antigen Kp90) have been used for the rapid diagnosis of TB. In this study, we report the isolation of an immunodominant lipopolysaccharide (LPS) antigen from M. tuberculosis H(37)Rv, which can be used for the serodiagnosis of TB. The LPS antigen was compared with three commercially available mycobacterium-specific antigens for the detection of TB. The antigens were evaluated using serum samples obtained from 59 Indian patients (19 patients with active pulmonary TB, 20 with extrapulmonary TB, and 20 with nontuberculous pulmonary disease) and 20 healthy adults. Antigen 60 IgG (sensitivity 89%, specificity 97%) and LPS (sensitivity 84%, specificity 97%) were more sensitive and specific than 38 kDa antigen IgG (sensitivity 79%, specificity 97%) and Kp90 IgA (sensitivity 82%, specificity 40%). These results indicate that the LPS antigen can be used as a sensitive tool for the serodiagnosis of TB and could be utilized to develop an ELISA for the screening of patients for TB.     

The role of solithromycin in the management of bacterial community-acquired pneumonia

The fluoroketolide solithromycin is 2-fold more potent in vitro than telithromycin against pneumococci (including macrolide-resistant strains) and Haemophilus influenzae and very active on pathogens causing atypical pneumonia. In contrast, it is a 30-fold less potent inhibitor of nicotinic receptors incriminated in telithromycin toxicity. In Phase II/III trials, oral solithromycin once-daily (800 mg on day 1; 400 mg on days 2-5) proved effective and safe when compared to respiratory fluoroquinolones for the treatment of community-acquired bacterial pneumonia (CABP). A Phase III intravenous trial vs. moxifloxacin has been recently completed for the same indication. Solithromycin may restore interest in ketolides as a first-line therapy for CAPB. Solithromycin safety should nevertheless be confirmed in larger populations allowing for detection of rare adverse events.     

耐碳青霉烯类革兰阴性菌肺炎的发生及死亡风险因素分析

目的 探讨影响耐碳青霉烯类革兰阴性菌（CR GNB）肺炎发生及其死亡的风险因素,为临床经验性诊治和预防提供参考。 方法 回顾性分析2016年1月至12月226例革兰阴性菌肺炎住院患者,根据药敏结果分为碳青霉烯类耐药组114例和非耐药组112例,根据预后将耐药组分为存活组88例和死亡组26例。采取单因素及多因素分析,确定引起CR GNB肺炎发生及死亡的独立危险因素。结果 单因素分析显示,入住ICU、近期手术、长期卧床、发热、意识障碍、90天内使用碳青霉烯类抗生素、呼吸衰竭、休克、感染前有创操作、感染病原菌（ABA、SMA）、院内获得,在耐药组的比例明显高于非耐药组;入住ICU、冠心病、糖尿病、肾功能不全、呼吸衰竭、休克、留置导尿、深静脉置管、机械通气,在死亡组的比例明显高于存活组。多因素分析显示,之前90天内应用碳青霉烯类抗生素以及机械通气、鲍曼不动杆菌感染、院内获得的患者发生CR GNB肺炎的风险增加。休克、深静脉置管、糖尿病、冠心病的CR GNB肺炎患者死亡风险增加。结论 之前90天内应用碳青霉烯类抗生素以及机械通气、鲍曼不动杆菌感染、院内获得是发生CR GNB肺炎的独立危险因素;休克、深静脉置管、糖尿病、冠心病是其死亡的独立危险因素。     

小儿肺炎克雷伯菌临床感染及耐药性分析

目的分析住院患儿肺炎克雷伯菌的临床分布及耐药性,以指导临床合理用药。方法对2009年10月至2010年12月儿科送检标本分离出的肺炎克雷伯菌的临床分布特点及耐药情况进行总结分析。结果共分离出肺炎克雷伯菌172株,其中产超广谱β-内酰胺酶(ESBLs)菌41株,占23.84%;主要来源于咽拭子、血液和痰液等,分别占70.93%、9.88%和8.14%;肺炎克雷伯菌对亚胺培南、厄它培南、丁氨卡那霉素和哌拉西林/他唑巴坦敏感性高。结论小儿肺炎克雷伯菌主要引起下呼吸道感染;对氨苄西林、氨苄西林/舒巴坦及头孢类抗菌药物耐药率高,哌拉西林/他唑巴坦和亚胺培南可作为治疗儿童产ESBLs肺炎克雷伯菌感染或非产ESBLs菌严重感染的首选用药。     

梅毒螺旋体Tp0453重组蛋白的表达及在梅毒血清学诊断中的初步应用

目的克隆、表达梅毒螺旋体（Tp）外膜蛋白Tp0453，建立间接ELISA法，探讨其在梅毒血清学诊断中的应用。方法构建重组质粒pQE32／Tp0453，IPTG诱导表达，SDS-PAGE和免疫印迹鉴定表达结果；Ni-NTA亲和层析法纯化重组蛋白，间接酶联免疫吸附试验（ELISA）检测人血清中特异性IgG抗体。结果成功构建了重组质粒pQE32／Tp0453；SDS-PAGE检测诱导产物显示有一分子量约为32000的特异蛋白带，免疫印迹检测其只与Tp IgG抗体具有阳性反应；重组蛋白用作ELISA包被抗原检测Tp阴阳性参比血清，特异度和灵敏度均为100％；检测患者血清中特异性IgG抗体结果与TPPA法符合率为98．2％。结论表达的Tp043重组蛋白具有较好的免疫反应活性，可望用于梅毒的血清学诊断。     

2000-2009年同济医院肺炎链球菌的耐药性分析

目的 了解肺炎链球菌的耐药情况及变化趋势.方法 收集同济医院2000年1月1日至2009年12月31日分离的753株肺炎链球菌,主要分离自呼吸道标本,其次为血液和脑脊髓液标本.采用E-test法检测分离株对青霉素及头孢曲松的MIC,其余采用纸片扩散法进行抗菌药物敏感性试验.结果 10年来总的非脑膜炎来源PNSSP占23.8%(93/392),儿童PNSSP占26.4%(47/178),成人PNSSP占16.8%(36/214),差异具有统计学意义(χ2=7.642,P＜0.01).10株脑膜炎标本来源肺炎链球菌均为PRSP.莫西沙星、左氧氟沙星对肺炎链球菌仍然保持着良好的敏感性,敏感率分别为96.9%(720/743)和90.5%(672/743),未发现万古霉素和美罗培南耐药菌株.肺炎链球菌对大多数抗菌药物的耐药性均有不同程度升高,尤其是青霉素、红霉素及克林霉素敏感性下降尤为明显.2006年PNSSP仅19%(19/99),2009年上升为30%(35/114);2000年红霉素敏感性为22%(28/125),至2009年仅为3%(3/114);2004年克林霉素的敏感性为40%(13/32),至2009年仅为4%(5/114).结论 2000-2009年同济医院分离的肺炎链球菌对青霉素、红霉素及克林霉素的耐药性呈上升趋势,尤其是儿童对此3种抗菌药物的耐药性明显高于成人分离株.临床应结合药敏试验结果合理选择抗菌药物.

Abstract：

Objective To investigate antibiotic resistance and resistant trend of Streptococcus pneumonia. Methods To investigate 753 Streptococcus pneumoniae isolated from Tongji Hospital in recent 10 years from January 1st 2000 to December 31st 2009, most of them were from respiratory tract specimens,followed by blood and cerebrospinal fluid. The MIC to penicillin & cefatriaxone were determined by E-test,and other antimicrobial susceptibility were tested by Kirby-Bauer method. Results For non-cerebrospinal fluid specimen, the total rate of PNSSP was 23.8%( 93/392 ), it was significant different between the rate of PNSSP from children ( 26. 4%, 47/178 ) and adults ( 16. 8%, 36/214, χ2 = 7. 642, P ＜ 0. 01 ). All of 10 strains isolated from cerebrospinal fluid were PRSP. Most isolates were high-susceptive to moxifloxacin and levofloxacin, and the rate of susceptibility were 96. 9% ( 720/743 ) and 90. 5% ( 672/743 )respectively. None of Streptococcus pneumonia was resistant to vancomycin and meropenam. The resistant rate of most tested antibiotics increased in different degree year by year, especially penicillin, erythromycin and clindamycin. The rate of PNSSP was only 19%( 19/99 )in 2006 ,but in 2009 the rate increased to 30%( 35/114 ). The susceptibility rate of erythromycin was 22% ( 28/125 )in 2000, but only 3% ( 3/114 )in 2009 ;and the susceptibility rate of clindamycin decreased from 40% ( 13/32 ) in 2004 to 4% (5/114) in 2009. Conclusions From 2000 to 2009, Streptococcus pneunoniae was more likely resistant to penicillin,erythromycin and clindamycin year by year, especially those isolates recovered from children. It was suggested that antibiotics should be chosen to use according to antimicrobial susceptibility test results.