儿童呼吸道感染者肺炎支原体及肺炎衣原体特异性抗体检测临床意义

目的 探究儿童呼吸道感染者肺炎支原体及肺炎衣原体特异性抗体检测临床意义。方法 选择我院2018年1月~8月收治的85例呼吸道感染患儿设为研究组,另选择同期在我院进行健康体检的85例儿童设为对照组,对照组采用ELISA法检测肺炎支原体及衣原体,研究组在此基础上行咽拭子培养法检测,比较两组肺炎支原体、衣原体抗体检出率,ELISA法与咽拭子培养法对肺炎支原体、衣原体抗体的阳性检出率,分析研究组呼吸道感染性疾病中肺炎支原体、衣原体抗体阳性分布情况。结果 研究组患儿肺炎支原体抗体MP-IgM抗体、MP-IgA抗体、MP-IgG抗体及肺炎衣原体特异性抗体CP-IgM抗体、CP-IgG抗体的检出率均高于对照组儿童,差异有统计学意义（P＜0.05）;ELISA法对肺炎支原体特异性抗体阳性的检出率为54.12%高于咽拭子培养法的36.47%,组间对比具有统计学意义（P＜0.05）;ELISA法对肺炎衣原体特异性抗体阳性的检出率为58.82%高于咽拭子培养法的37.65%,差异有统计学意义（P＜0.05）;肺炎支原体、肺炎衣原体抗体阳性在各种儿童呼吸道感染性疾病中均有表现,其中,肺炎和支气管肺炎的阳性检出率较咽炎、扁桃体炎均较高。结论 肺炎支原体、肺炎衣原体是引发肺炎和支气管肺炎感染的主要病原菌。     

套式（Nested）PCR检测肺炎支原体与肺炎衣原体研究

本文报告根据肺炎支原体（Mp）和肺炎衣原体（Cpn）的16SrRNA基因序列，建立了套式（Nested）PCR检测方法。灵敏度试验表明，Mp套式PCR和Cpn套式PCR灵敏度分别高出Mp普通PCR和Cpn普通PCR二个数量极。     

肺炎支原体抗原酶联免疫检测方法的建立

目的采用ELISA技术,建立肺炎支原体（Mycoplosma pneumonia）抗原检测方法。方法制备肺炎支原体的多克隆抗体,建立酶联免疫吸附法,检测标本中的肺炎支原体,对其敏感性和特异性进行检测。结果本方法可以检测毒株的1／2^12的稀释样品,对甲型流感病毒（H3N2亚型、H1N1亚型）、乙型流感病毒、副流感病毒（Ⅰ型、Ⅲ型）、呼吸道腺病毒（Ⅲ型、Ⅶ型）、肺炎衣原体无特异性反应。结论本肺炎支原体抗原检测方法可用于疑似肺炎支原体感染样品的临床诊断和鉴别诊断。     

冠心病患者肺炎衣原体DNA及抗体检测

目的:探讨肺炎衣原体(Cpn)与冠状动脉粥样硬化心脏病(CHD)之间的相互关系。方法:采用PCR和ELISA ,分别对2 0 0例经冠状动脉造影证实的冠心病患者和非冠心病对照组进行全血标本肺炎衣原体DNA和血清中抗肺炎衣原体IgG检测。结果:冠心病组肺炎衣原体DNA阳性率为4 3 4 0 % ,对照组阳性率为7 32 %。冠心病组肺炎衣原体IgG抗体阳性率为6 4 78% ,对照组阳性率为31 71%。两组肺炎衣原体DNA和IgG抗体阳性率差异均有显著性(P <0 0 1)。结论:肺炎衣原体感染与冠心病存在密切的关系     

儿童肺炎肺炎支原体及肺炎衣原体联合基因诊断研究

目的：探讨我国肺炎儿童中肺炎支原体（Mycoplasma pneumoniae,Mpn）和肺炎衣原体（Chlamydia pneumoniae,Cpn）的感染状况和Mpn和Cpn合并感染状况。方法：收集80例健康儿童和134例肺炎儿童之咽拭子标本,应用套式PCR（nested PCR）技术进行检测,并随机抽取Mpn和Cpn和3例套式PCR阳性产物进行全自动荧光DNA测序确诊。结果：肺炎儿童Mpn和Cpn套式PCR阳性检出率分别高达30.0%和35.0%,明显高于健康儿童的2.5%和2 .5%（P均＜0.01）,肺炎儿童中Mpn、Cpn双重阳性者为27例,达20.1%。结论：Mpn和Cpn是我国儿童肺炎的重要病原体,并存在较高比例的Mpn、Cpn双重感染者。     

肺炎衣原体特异性单克隆抗体的研制及应用

采用肺炎衣原体(Chlamydia pneumoniae, Cpn)TW-183的纯化原体免疫BALB/C雄性小鼠, 将小鼠脾脏细胞与SP2/0细胞融合, 杂交细胞通过有限稀释法克隆, 获得1株稳定分泌Cpn-单克隆抗体(Cpn-McAb)的细胞株, 单抗属IgG2b亚型及抗Cpn-MOMP抗体.自制Cpn-McAb与鹦鹉热衣原体原体有较弱的交叉反应; 与沙眼衣原体原体无交叉反应, 特性与进口Cpn-McAb一致.为了评估Cpn-McAb, 用自制和进口Cpn-McAb同时检测外周血单核细胞标本454例, Cpn-Ag阳性率分别为53.3%和52.6%, Kappa值0.714, 两者有较高的一致性.结果提示,自制Cpn-McAb几乎和进口Cpn-McAb一样有较高的特异性和敏感性,可替代进口单抗用于临床Cpn感染的检测, 有助于相关疾病的诊治.     

肺炎支原体抗体联合超敏C反应蛋白检测在小儿支原体肺炎感染诊断中的临床价值

目的 调查研究小儿支原体肺炎感染患者肺炎支原体抗体(MP-Ab)及超敏C反应蛋白(hs-CRP)水平的变化,探讨肺炎支原体抗体与超敏C反应蛋白联合检测对小儿支原体肺炎感染的临床诊断价值.方法 肺炎支原体肺炎患儿110例,对照儿童120例,均按照《诸福棠实用儿科学》中支原体肺炎诊断标准确诊,两组分别进行MP-Ab、hs-CRP检测,肺炎支原体抗体判定以MP-Ab滴度≥1:160为阳性,超敏C反应蛋白判定以hs-CRP＞ 5mg/L为阳性.通过分析MP-Ab检测法与MP-Ab联合hs-CRP检测法敏感性、特异性差异,探讨MP-Ab联合hs-CRP检测法诊断小儿支原体肺炎感染的临床意义.结果 肺炎支原体肺炎患儿组MP-Ab滴度≥1∶160比率高于正常对照组,肺炎支原体肺炎患儿组hs-CRP高于正常对照组,差异均有统计学意义(P＜0.05);MP-Ab检测法诊断肺炎支原体肺炎的灵敏度为78.2％,MP-Ab联合hs-CRP检测法诊断肺炎支原体肺炎的灵敏度为89.1％,MP-Ab联合hs-CRP检测肺炎支原体肺炎的灵敏度高于MP-Ab检测法,差异有统计学意义(P＜0.05);MP-Ab检测法诊断肺炎支原体肺炎的特异度为94.2％,MP-Ab联合hs-CRP检测法诊断肺炎支原体肺炎的特异度为89.2％,差异无统计学意义(P＞0.05).结论 MP-Ab与hs-CRP联合检测能提高小儿支原体肺炎感染的诊断率,具有较高临床价值.     

PCR检测肺炎患儿咽拭子肺炎支原体

本文应用ＰＣＲ技术检测了２７５例肺炎患儿之咽拭子标本中的肺炎支原体特异性ＤＮＡ，结果为７１例患儿之咽拭子阳性，阳性率２５．８％，与国内其他作者的报道相近。本文并就肺炎支原体感染的特异性诊断方法作了复习与讨论。     

支气管肺泡灌洗液与咽拭子荧光定量PCR法检测成人肺炎支原体感染的对比研究

目的 用实时荧光定量PCR方法对肺部感染危重患者的咽拭子样本和支气管肺泡灌洗液（BALF）进行检测,探讨不同样本对检测结果的影响.方法 450例肺部感染危重患者纳入临床研究,用实时荧光定量PCR方法检测患者咽拭子样本和BALF样本,对检测结果进行比较.结果 在450例肺部感染危重患者中,肺炎支原体肺炎（MPP）患者1 15例,占25.6％.BALF样本检测的阳性率为69.56％,咽拭子样本检测的阳性率为13.04％.肺炎支原体（MP）感染的年龄段主要在20～39和50 ～59岁,以50-59岁年龄段人数最多.结论 MP是肺部感染主要病原体之一,采集BALF标本用实时荧光PCR检测MPP阳性率更高,是MPP诊断的有效手段.     

儿科感染患者的肺炎衣原体检测研究

肺炎衣原作是一种专性细胞内寄生微生物，已构成肺炎的第3位或第4位致病源。本文应用灵敏、特异的套式PCR技术、检测了81例儿科感染患者的咽拭子标本的肺炎衣原体特异性DNA，总阳性率13．6％。     

肺炎支原体感染在BALB/c小鼠肺组织不同时间炎症的变化

目的了解肺炎支原体感染的不同时期肺组织的病理变化、用PCR的方法检测肺炎支原体出现时间、阳性率的变化情况。方法建立肺炎支原体经呼吸道感染的BALB/c小鼠模型,对感染后3、7、14、21 d的小鼠的肺组织进行病理变化、肺湿重、PCR检测肺炎支原体出现时间的研究。结果肺炎支原体经呼吸道感染的BALB/c小鼠肺组织变大重量增加,肺指数同正常对照组相比,P﹤0.01,差异有统计学意义;呼吸道感染的BALB/c小鼠肺组织在3、7 d时,肺组织的病理变化最明显,14 d炎症减轻,21 d炎症基本消退;用PCR的方法检测支气管肺泡灌洗液肺炎支原体7 d时全部为阳性。结论肺炎肺炎支原体感染BALB/c小鼠模型制作成功,感染后7 d时肺部炎症最明显。     

Recombinant 46-kilodalton surface antigen (P46) of Mycoplasma hyopneumoniae expressed in Escherichia coli can be used for early specific diagnosis of mycoplasmal pneumonia of swine by enzyme-linked immunosorbent assay.

The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae. Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique. The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system. The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M. hyopneumoniae in swine serum. It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae. With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection.     

一例肠道腺病毒41型感染与重症肺炎关系的临床分析

目的分析肠道腺病毒与呼吸系统疾病的关系，进一步提高婴幼儿重症肺炎诊治水平。方法对重症肺炎尸检脑、肝、心、肺、肠、脾组织进行腺病毒巢式PCR检测，对PCR产物进行克隆测序：同时用免疫组织化学方法检测肺组织中3型腺病毒、支原体、衣原体、呼吸道合胞病毒、A型流感病毒、B型流感病毒的分布情况。结果免疫组织化学结果表明3型腺病毒、支原体、衣原体、呼吸道合胞病毒、A型流感病毒、B型流感病毒都为阴性。肺和肠中腺病毒巢式PCR结果为阳性，测序结果显示都为腺病毒41型。结论肠道腺病毒41型感染可能是婴幼儿重症肺炎的重要病因之一。     

Diagnostic Relevance of the Detection of Legionella DNA in Urine Samples by the Polymerase Chain Reaction

 Urine samples from 317 patients with pneumonia and from 242 patients without pneumonia were tested using a polymerase chain reaction (PCR) system for detection of the Legionella 5S rRNA gene. The results were compared with findings obtained using the established methods for diagnosis of legionellosis. Of the 317 patients with pneumonia, 58 had confirmed legionellosis, 35 had a presumptive Legionella infection, and 224 had no evidence of Legionella infection as determined by conventional methods using published criteria. The PCR was positive for 42 patients with confirmed infections, yielding a sensitivity of 72.4%. Furthermore, 16 (47%) patients with presumptive legionellosis and five (2.2%) patients without other evidence of Legionella infection had positive results. All samples from 242 patients without pneumonia were PCR-negative. When the results for all patients were considered, the specificity of the assay was ≥98.9%. The results demonstrate that the sensitivity and specificity values of urinary PCR are in the same range as those of established methods. The use of PCR in urine complements the repertoire of rapid diagnostic methods, especially for infections caused by legionellae not belonging to Legionella pneumophila serogroup 1, in which tests for detection of urinary antigen often fail.     

人疱疹病毒7型被膜蛋白pp85的原核表达及应用

目的构建人疱疹病毒7型(HHV7)被膜蛋白pp85编码基因(ORF U14)的原核表达载体,并进行原核表达。方法应用HHV7标准株Glasgow感染SupT1细胞,PCR技术扩增HHV7ORFU14基因的主要抗原决定簇编码区(328~533AA),目的基因与原核表达载体pThioHis A连接后,转化宿主菌E.coliBL21,IPTG诱导融合蛋白表达,经镍-螯合物琼脂糖树脂柱亲和层析纯化获得重组抗原。重组蛋白电泳后转至PVDF膜,与HHV7阳性血清进行免疫印迹反应。结果DNA序列分析表明,目的基因序列与HHV7标准毒株Glasgow相应序列完全一致,SDS-PAGE可观察到相对分子质量(Mr)为35.7×103融合蛋白的表达,免疫印迹反应显示重组抗原具有较高的特异性。结论HHV7重组抗原具有较好的抗原性,进一步完善后可用于HHV7抗体的检测。     

Species-specific monoclonal antibodies to Escherichia coli-expressed p36 cytosolic protein of Mycoplasma hyopneumoniae

The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.     

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.     

An open, randomized, comparative study of clarithromycin and erythromycin in the treatment of children with community-acquired pneumonia.

BACKGROUND AND PURPOSE: This study aimed to evaluate the efficacy and safety of clarithromycin and erythromycin in the treatment of community-acquired pneumonia in children. METHODS: Children with community-acquired pneumonia were randomly assigned to receive 10-day regimens of either clarithromycin 15 mg/kg/day, twice a day, or erythromycin 30-50 mg/kg/day, four times daily. RESULTS: A total of 97 children entered this study, including 26 with Mycoplasma pneumoniae infection, 15 with Chlamydia pneumoniae infection, and 6 with mixed mycoplasma and chlamydia infections. Fifty and 47 children received clarithromycin and erythromycin treatment, respectively. Three children withdrew from the study because the identified pathogens were resistant to the study drugs. All 47 children with mycoplasma or chlamydia infection were cured clinically. Delayed defervescence, defined as a fever lasting for more than 72 h after treatment, was observed in 4 of 22 clarithromycin-treated children (18%) and in 3 of 15 erythromycin-treated children (20%) [p>0.05]. Gastrointestinal side effects, including vomiting, abdominal pain and diarrhea, were observed in 3 of 50 children (6%) receiving clarithromycin and in 11 of 49 children (22%) receiving erythromycin (p=0.039). Excluding children with abnormal pretreatment liver function, abnormal liver function after treatment was observed in only one child, treated with erythromycin. Post-treatment eosinophil and platelet counts were significantly elevated after treatment in both groups. CONCLUSIONS: Clarithromycin showed efficacy equivalent to erythromycin for the treatment of mycoplasma or chlamydia pneumonia in children. However, the tolerability of clarithromycin was superior to that of erythromycin.     

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZαA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 °C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2–100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries.