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.     

支气管肺泡灌洗液细胞学诊断肺泡蛋白沉着症1例

患者女性 ,43岁 ,工人。因慢性咳嗽、咳痰、进行性呼吸困难 4年余 ,加重伴低热 1年入院。患者既往有高分子粘合剂粉尘接触史 11年 ,油漆接触史 7年。胸部Ｘ线示 :两肺下野可见肺纹理增粗并连成网状 ,右下外带呈毛玻璃状改变 ,心肋膈角锐利。胸部ＣＴ示两肺中下肺野可见分布不均匀的片状毛玻璃样病灶。肺功能检查 :提示肺通气功能中度障碍。病理检查　送检支气管灌洗液标本 ,标本底部肉眼可见米黄色淤泥样沉积物。涂片镜下所见 :在纤毛柱状上皮细胞、炎细胞及少许吞噬细胞背景中 ,可见均匀粉染无结构球形小体 ,直径 2 0～ 5 0 μｍ ,球形小体…     

支气管肺泡灌洗液诊断肺结核的诊断的价值应用

目的评价纤维支气管镜防污染支气管肺泡灌洗液技术对肺结核分支杆菌快速培养和痰菌阴的不典型肺结核的诊断价值应用。方法对98例肺结核患者采用支气管镜进行检查,并对病变部位灌洗标本液,并且培养出结核杆菌。结果98例结核分支杆菌检出率为66.3%、84.8%,其中30例活检,20例确诊为结核内芽肿,检出率为66.7%。结论纤支镜下进行BALF快培对不典型肺结核具有良好的诊断及时,阳性率较高,通过支气管镜下不同的取材方法能够有效的提高诊断率。     

122例重症肺炎患儿支气管肺泡灌洗液的病毒检测分析

目的 了解湖南长沙地区重症肺炎患儿中呼吸道病毒的感染状况.方法 2011年1月至2011年12月,收集重症肺炎儿童的肺泡灌洗液标本122份,采用巢氏或普通逆转录聚合酶链反应(nested-PCR或RT-PCR)对腺病毒(ADV)、博卡病毒(HBoV)及副流感病毒1、2和4(PIV1、2、3、4)、呼吸道合胞病毒(RSV)、鼻病毒(HRV)、流感病毒A、B(IFVA-B)、人偏肺病毒(HMPV)、冠状病毒HKU1及NL63(HCoV-HKU1、HCoV-NL63)进行检测.结果 122份重症肺炎患儿肺泡灌洗液标本中,60份病毒阳性(49.1％),其中ADV检出率最高(40.98％,50/122),其次为RSV (7.37％,9/122),HBoV(7.37％,9/122).2种病毒混合感染有21例,占35％ (21/60),40％(20/50)的ADV阳性存在混合感染.结论 2011年,腺病毒感染是长沙地区重症肺炎的重要病因,其中腺病毒的混合感染在重症肺炎中常见.     

吸烟致气道炎症反应早期诊断方法的探讨

目的 寻找临床上简便易行、可靠的早期诊断吸烟致气道炎症的方法.方法 检测了51例健康吸烟者与46例健康不吸烟者支气管肺泡灌洗液(BALF),并以BALF中细胞计数绝时值将炎症程度分为Ⅰ、Ⅱ、Ⅲ度;同时进行乙酰甲胆碱(Mch)支气管激发试验与24h最大呼气流量(PEF)测定;结果①51例健康吸烟者BALF细胞计数绝对值为(2.3～47.2)×108/L,其炎症分度为Ⅰ度16例、Ⅱ度22例、Ⅲ度13例.46例健康不吸烟者炎症分度均为Ⅰ度.②Mch支气管激发试验51例健康吸烟者阳性率64.7%.(Ⅰ度50%、Ⅱ68.2%、Ⅲ度76.9%),46例健康不吸烟阳性率15%.③24h-PEF变异值异常率为62.7%(Ⅰ度37.5%、Ⅱ72.7%、Ⅲ度76.7%).以结果与BALF炎症程度的相关性较好.结论 本文方法可较好地反映吸烟致气道炎症的严重程度,Mch支气管激发试验与PEF测定可作为吸烟气道炎症早期诊断的无创性指标之一.     

支气管肺泡灌洗液CEA和CA-50测定对肺癌的诊断价值

流行病学调查发现肺癌发病率在逐年升高，并且绝大多数肺癌病人在临床确诊对已属中，晚期。因此，及时发现早期病例对于肺癌的治疗具有积极的意义。本文应用放射免疫分析方法测定了肺癌和肺良性疾病患者支气管肺泡灌洗液(BALF)中CEACA-50含量，并与其血清水平和阳性率进行了比较，以探讨BALF检测对肺癌早期诊断的临床价值。     

支气管肺泡灌洗液在鉴别肺良恶性肿瘤中的价值

本文对54例肺疾病患者的肺泡灌洗液及血清进行了CYFRA21-1、CEA水平检测,其中肺良性疾病21例,肺癌33例,同时检测健康人血清41例并加以比较,发现它对早期鉴别肺恶性肿瘤可提供有价值的依据,现报道如下. 1 对象和方法 1.1 对象 `1.1.1 对照组 41例(男31,女10),年龄(31～59)岁,为我院体检中心确认的健康者,各项指标正常,无呼吸、肝、肾、肺等重要器官疾病. 1.1.2 疾病组 54例(男33,女21),年龄(45～71)岁,为我院及另一医院呼吸科住院病人,经临床病理学、细胞学及影像学检查确诊肺癌32例,其中鳞癌17例、腺癌14例、小细胞癌1例.肺良性疾病22例(男14,女8),年龄(32～66)岁.     

支气管肺泡灌洗液检测CEA,CYFRA21—1对肺癌诊断价值的探讨

本文旨在探讨检测支气管肺泡灌洗液(BALF)中CEA、CYFRA21—1水平对肺癌的诊断价值。 资料和方法 一、临床资料:肺癌组32例(男26,女6),年龄43～86岁,平均63岁。均经胸片、胸部CT、纤支镜检查确诊并有病理检查证实。其中鳞癌7例,腺癌6例,肺泡细胞癌1例,鳞腺癌2例,不定型16例。对照组30例(男18,女12),年龄43～71岁,平均58岁,为同期住院病人,亦经胸片、胸部CT、纤支镜检查确诊。其中支气管扩张伴咯血12例,肺炎6例,间质性肺炎2例,支气管炎6例,肺结核1例,慢支咯血3例。     

哮喘患者动脉血及支气管肺泡腔灌洗液中内皮素—1的浓度

内皮素是一个具有很多生物学特性的缩氨酸介质家族。其中包括收缩气道和血管的作用。内皮素 - 1(ET- 1)比同家族的其余两种缩氨酸更有效。为了了解 ET- 1在哮喘发病机理中的作用 ,我们检测了 11位遗传性过敏性哮喘患者发作期和缓解期以及 11位健康对照者的动脉血中 ET- 1水平 ,并通过纤维支气管镜对 11位缓解期哮喘患者和 11位健康对照者行支气管灌洗术以检测支气肺泡腔灌洗液 (BAL )中 ET- 1水平。动脉血及 BAL中的 ET- 1浓度用放免法测得。动脉血 ET- 1水平不论在哮喘持续组 (3.6 7± 0 .5 1pg/ml)或缓解组 (2 .6 5± 10 .6 2 pg/…     

支气管哮喘小白鼠支气管肺泡灌洗液中TGF-β含量的变化

气道重构 (airwayremodeling)是难治性支气管哮喘气道发生不可逆性气流阻塞及持续性非特异性气道高反应性的重要原因之一。TGF β是分子量约 2 5kD的蛋白质 ,最近报道TGF β具有刺激纤维母细胞促进纤维结合素及胶原的等间质成分的产生[1 ] ,于是推测TGF β在支气管哮喘气道重构中可能起重要的作用。在国内定量性研究TGF β在支气管哮喘气道重构中的作用尚无文献报道。本实验用小白鼠建立支气管哮喘气道重构的动物模型测定 (bronchoalveollarlavageflu id ,BALF)中TGF β的含量阐明气道重构中它的作用。1　材料与方法1 1　动物 :动物…     

Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the detection of the DeltaF508 mutation

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.     

双重荧光PCR检测HIV前病毒DNA及其在婴幼儿HIV感染早期诊断中的应用

目的建立双重荧光PCR检测HIV前病毒DNA的方法,并应用于婴幼儿HIV感染的早期诊断。方法采用TaqMan技术,组建针对人类核糖核酸酶P（ RNase P）和HIV的长末端重复序列（ LTR）基因的双重荧光PCR体系;采用TA克隆技术构建pTG19-T重组质粒作为模板进行该方法灵敏度的评价;采用11例已知健康人的血样和98例已知HIV感染者血样进行该方法的特异性验证;收集2011年1月至2012年9月浙江省各地妇幼保健医疗机构上送的96份婴幼儿样本,用新方法进行HIV的早期诊断,并将检测结果与罗氏HIV DNA定性检测试剂盒作比较。结果双重荧光PCR新方法能特异性检测HIV前病毒DNA,特异性为100％,检测灵敏度为100拷贝/反应;新方法和罗氏HIV DNA定性检测试剂盒检测96份婴幼儿样本,结果完全一致（符合率为100％）。结论建立的双重荧光PCR方法经济便捷、特异性好、灵敏度高、结果准确、易于推广,有望用于婴幼儿HIV早期诊断,并为HIV前病毒DNA的检测提供了一个通用技术平台。     

双重引物PCR鉴定APPSWE转基因小鼠

本实验旨在探讨一种简单易行,能有效识别APPSWE转基因小鼠基因检测中假阴性结果的方法。在PCR体系中设计两对引物,一对为APP基因特异性引物,另一对为根据其内源性管家基因β-actin设置的参照引物。利用同一个PCR反应体系,对两对引物进行扩增。结果显示,双重引物PCR扩增的特异性片段与单引物扩增片段完全吻合,利用该法检出的转基因阳性率高于单引物PCR检出的转基因阳性率。本方法简单易行,能够有效识别以往利用单一PCR检测出现的假阴性结果,并且具有很好的特异性和灵敏性,值得在转基因小鼠鉴定实验中推广。     

Evaluation of nested and real-time PCR assays in the diagnosis of candidaemia

Polymerase chain reaction (PCR) assays have a very low theoretical detection threshold and are therefore advocated for the diagnosis of fungaemia. However, their effectiveness in this respect remains to be assessed. This study compared real-time PCR ( Can -G) and nested PCR assays with blood culture for the diagnosis of Candida spp. bloodstream infections. A total of 200 clinical blood samples obtained from 110 patients at risk for developing a systemic fungal infection, hospitalized in the University Hospital of Sfax (Tunisia), were submitted to testing by culture, nested PCR and real-time PCR. Blood culture was positive in 36 patients. When compared with culture, the Can -G assay (81% sensitivity, 96% specificity) performed better than the nested PCR assay (86% sensitivity, 54% specificity). The real-time PCR assay, which avoids both the contamination hazard with amplicons that may cause false-positive results and the use of time-consuming post-PCR steps, appears more suitable than the nested PCR assay for the laboratory diagnosis of Candida spp. bloodstream infections. In this study, real-time PCR did not enhance the diagnostic sensitivity for Candida spp. bloodstream infections compared with conventional blood culture; however, it may lead to earlier implementation of an adequately targeted antifungal treatment.     

Improvement of pneumococcal pneumonia diagnosis using quantitative real-time PCR targeting lytA in adult patients: a prospective cohort study

ObjectivesThe aim of the study was to assess the performance of real-time PCR targeting the lytA gene (rtPCR-lytA) in plasma, urine and nasopharyngeal (NP) samples for the diagnosis of pneumococcal community-acquired pneumonia (P-CAP).MethodsProspective observational study including all consecutive adults with CAP from November 2015 to May 2017. P-CAP was defined if pneumococcus was identified using conventional methods (CM) and/or a positive rtPCR-lytA was detected in blood, urine or NP samples (NP cut-off ≥8000 copies/mL). Diagnostic performance of each test was calculated.ResultsA total of 133 individuals with CAP were included. Of these, P-CAP was diagnosed in 62 (46.6%). The proportion of P-CAP diagnosed by rtPCR-lytA methods was significantly higher than that diagnosed by CM (87.1% versus 59.7%, p 0.005). The rtPCR-lytA identified Streptococcus pneumoniae in 25 patients (40.3% of all individuals with P-CAP) whose diagnosis would have been missed by CM. NP-rtPCR-lytA allowed diagnosis of 62.3% of P-CAP. A nasopharyngeal colonization density ≥2351 copies/mL predicted P-CAP diagnosis (area under the curve = 0.82, sensitivity 83.3%, specificity 80.9%). There was a positive correlation between increasing bacterial load in blood and CURB-65 score (Spearman correlation coefficient r = 0.4, p 0.001), pneumonia severity index (r = 0.3, p 0.02) and time to clinical stability (r = 0.33, p 0.01). Median bacterial load in blood was higher in P-CAP patients with bacteraemia (0.65 × 10³ versus 0 × 10³ copies/mL, p 0.002), intensive care unit admission (0.68 × 10³ versus 0 × 10³ copies/mL, p 0.04) or mechanical ventilation (7.45 × 10³ versus 0 × 10³ copies/mL, p 0.04).ConclusionsThe use of rtPCR-lytA methods significantly increased the diagnosis of P-CAP compared with CM. Nasopharyngeal swabs rtPCR-lytA detection, with an accurate cut-off value, was the most promising among molecular methods for the diagnosis of P-CAP.     

Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay

Objective   To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens.
Methods   A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii , Borrelia garinii and Borrelia valaisiana . A recombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard.
Results   The specificity of the assay was documented with 48 different clinically relevant Borrelia burgdorferi strains. No cross-reaction occurred with unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed. The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence. Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated. None of these samples showed measurable amounts of Borrelia burgdorferi DNA.
Conclusion   By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.     

Buffy coat PCR for diagnosis of experimental pneumococcal pneumonia

An immunocompetent murine model of pneumococcal pneumonia and bacteremia was used to evaluate a PCR assay based on amplification of the pneumolysin gene. Mice were treated with trovafloxacin to determine the decline in sensitivity of PCR as lung bacterial concentrations decreased and blood cultures became sterile. Forty-three mice were studied for up to 120 h after start of antibiotic treatment. PCR of buffy coat specimens was more sensitive than PCR of plasma. Only 21% of animals had a positive blood culture, whereas 77% of PCR buffy coat assays were positive. After 48 h of therapy all blood culture specimens were sterile, whereas buffy coat PCR was positive in 57.8% of specimens. PCR of buffy coat specimens was negative in all mice colonized nasally with Streptococcus pneumoniae and in rabbits with Escherichia coli bacteremia. Our results demonstrate that our PCR technique using buffy coat specimens is highly specific for invasive pneumococcal disease and remains positive in the majority of animals for at least 48 h after start of antibiotic therapy.     

术后早期炎性肠梗阻23例诊治分析

目的 探讨术后早期爽性肠梗阻的临床特点、诊断、治疗和预防。方法 对23例早期炎性肠梗阻的治疗结果进行回顾性分析。结果 23例患者经胃肠减压、应用生长抑素、肠外营养、肾上腺皮质激素等疗法治愈，平均95d，无一例2次手术。结论 术后早期炎性肠梗阻常发生于术后4～11d，有典型的临床特点，保守治疗可治愈。     

Usefulness of real-time PCR for lytA,ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.     

Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples

A real-time LightCycler assay for Legionella pneumophila was evaluated with 120 water samples potentially contaminated with PCR inhibitors. Results were obtained within five hours, with a detection limit equivalent to 800 cells/L. However, 11 of 22 culture-positive samples containing < 100 CFU/L were also positive by LightCycler assay, indicating the presence of significant numbers of non-viable cells. Following extraction, amplification inhibitors remained in four culture-positive samples, but only one contained > 800 CFU/L. The assay seemed suitable for rapidly screening large sample numbers for heavy contamination with L. pneumophila , but conventional culture should continue to be used to detect low contamination levels.