qPCR检测结核杆菌在肺和胸膜上皮样肉芽肿性疾病诊断中的意义

目的探讨荧光定量聚合酶链反应(Real-time polymerase chain reaction,q PCR)法在诊断及鉴别诊断肺和胸膜结核病和其他肉芽肿性疾病中的意义。方法收集经病理学检查发现上皮样肉芽肿性病变的肺和胸膜活检的石蜡包埋组织142例,应用q PCR法检测结核分枝杆菌DNA,同时行抗酸染色,并与临床资料对比进行回顾性分析。结果在142例肺胸膜活检标本中,60例临床诊断确诊为结核病。采用q PCR法检测结核分枝杆菌DNA阳性者58例,敏感性为96.67%,特异性为100%;抗酸染色阳性者17例,敏感性为28.33%,特异性为97.56%,差异有统计学意义(χ~2=21.47,P0.001)。结论结核分枝杆菌核酸检测有助于在石蜡包埋的肺和胸膜小活检组织中快速、准确的诊断结核病,可用于疑似结核的肉芽肿性病变的鉴别诊断。     

优化改良PCR法检测石蜡组织标本中结核分枝杆菌

正临床病理活检中肺外器官结核要多于肺结核~([1]),而病理学诊断是肺外结核病确诊的重要手段。结核病组织标本有其特征性的病理学形态特点,病理诊断医师可以怀疑其结核病变,但确诊需依靠抗酸杆菌染色等结果。但实践中抗酸杆菌染色等阳性率低,漏检率高~([2])。随着分子生物学检测技术在病理学诊断中的应用,部分大型医院已经应用荧光PCR     

荧光PCR法检测石蜡标本中结核分枝杆菌的流程改进

<正>结核分枝杆菌的检测对于有肉芽肿性炎特征组织病变的病理诊断具有重要的参考意义。目前,病理科常用的检测方法有基于细菌学的抗酸染色、金胺O荧光染色,基于分子病理学的PCR、Xpert MTB/RIF检测^[1-2]等。其中荧光PCR技术具有敏感性高、特异性好、速度快,且价格实惠的特点,被广泛应用于大中型医院病理科福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded,FFPE）组织的结核分枝杆菌的检测中^[3-6]。由于石蜡组织标本中的病菌分布不稳定,     

PCR-膜芯片检测石蜡包埋组织中结核杆菌耐药基因突变

目的探讨PCR-膜芯片技术检测石蜡包埋组织中结核杆菌耐药基因突变的可行性及其在临床病理中的应用价值。方法采集100例石蜡包埋的肺或淋巴结组织标本(临床诊断为结核患者80例,非结核患者20例),TB膜芯片检测标本中的结核杆菌IS6110基因,阳性者利用12对特异性引物进行多重PCR,扩增产物与含有59个探针的耐药-TB膜芯片反向点杂交检测结核杆菌耐药基因突变并测序验证。结果 TB膜芯片检测石蜡包埋组织标本中的结核杆菌IS6110基因,以临床诊断为标准时,灵敏度为52.5%(42/80),特异度为100%(20/20);以抗酸染色结果为标准时,灵敏度为90.5%(19/21),特异度为61.0%(36/59)。耐药-TB膜芯片检测石蜡包埋组织结核杆菌耐药基因突变,在42例结核杆菌IS6110基因阳性的标本中,检出INH基因突变2例,RFP或SM基因突变各1例,INH、RFP和EMB基因同时突变1例,与测序结果基本符合,提示上述5例可能为耐药结核病。结论应用PCR-膜芯片技术可提高石蜡包埋组织中结核杆菌基因的检出率,耐药-TB膜芯片检测结果可为耐药结核病的病理学诊断提供有价值的参考。     

应用荧光定量PCR法检测病理组织中幽门螺旋杆菌感染

目的探讨荧光定量PCR方法检测病理组织中幽门螺旋杆菌（Hp）的效果,为临床Hp感染诊断提供快速有效的检测方法。方法应用荧光定量PCR法及苯胺蓝染色法分别对106例临床送检胃窦黏膜组织标本进行幽门螺旋杆菌检测,对检测结果进行对比分析。结果在106例胃窦标本中,荧光定量PCR法检测阳性例数68例,阳性率为64．2％,苯胺蓝染色法检测阳性例数46例,阳性率为43．4％。两种方法检测阳性率比较有显著性差异（P〈0．05）。结论荧光定量PCR技术用于检测病理组织中幽门螺旋杆菌,具有灵敏度高、特异性强的特点,对确诊胃镜活检标本中幽门螺旋杆菌感染具有重要应用价值。     

荧光定量PCR技术检测结核DNA在不同类型样本中的应用

目的 探讨荧光定量PCR技术检测结核DNA在不同类型样本中的阳性率。方法 收集2016年1月~2018年3月在我院进行结核DNA检测的9020例患者,运用荧光定量PCR技术检测不同类型的样本。结果 9020例患者中,检测结果阳性1220例,总阳性率13.53%;痰液检测5688例,检测结果阳性741例,总阳性率13.03%;肺泡灌洗液检测1799例,检测结果阳性363例,总阳性率20.20%;脑脊液检测704例,检测结果阳性40例,总阳性率5.68%;胸水检测559例,检测结果阳性35例,总阳性率6.26%;腹水检测62例,检测结果阳性6例,总阳性率9.68%;尿液检测193例,检测结果阳性35例,总阳性率18.13%;心包积液检测15例,检测结果阳性0例,总阳性率0。结论 荧光定量PCR技术检测结核DNA具有特异、灵敏、快速、准确等特点,但临床医生应根据症状体征等选择合适的临床标本进行结核DNA的检测,将进一步提高阳性率,明确诊断。     

提高抗酸染色阳性率的几点细节

正在临床病理诊断中遇到肉芽肿性病变伴干酪样坏死或结核样结节常常应用抗酸染色作诊断与鉴别诊断。作为基层医院,在没有荧光显微镜无法进行金胺O染色且无条件开展PCR定量检测技术的实验室,Ziehl-Neelsen苯酚碱性品红法检测抗酸杆菌是最常用也是最传统、经典的染色法,由于其成本低,操作简单、直观准确、费用少且无需专门仪器设备,在病理实验室中广泛应用,但由于该方法特异性高、阳性率较低,为了提高抗酸杆菌的检出率,作者对染液的配制、染     

智能化抗酸杆菌检测在结核诊断中的应用

目的 探讨智能化抗酸杆菌检测在结核病理诊断中的应用。方法 收集150例肉芽肿性改变的石蜡包埋组织,同时进行手工抗酸染色和自动抗酸染色,分别进行人工阅片病理诊断。使用自主开发的结核杆菌识别人工智能(tuberculosis bacillus finder, TB-Finder)作为初筛,另请病理医师复核150张自动抗酸染色切片并作出病理诊断,分析两种染色模式的效率及准确率。结果 150例智能化平台染色的阳性率为29.3%(44/150),传统模式染色的阳性率为6.6%(10/150)。染色加诊断全流程智能化平台每张切片耗时12 min,传统模式每张切片耗时57 min,智能化平台节约时间成本约78%。结论 智能化抗酸杆菌检测平台的应用可提高病理医师的工作效率和诊断准确率,有助于规范质量控制。     

EDTA修复肉芽肿组织中荧光定量PCR检测结核杆菌的应用

目的观察EDTA热修复炎性肉芽肿组织石蜡切片,能否提高荧光定量PCR结核/非结核分枝杆菌的检出率。方法对125例炎性肉芽肿组织石蜡切片结核/非结核分枝杆菌同时进行常规荧光定量PCR检测和EDTA热修复后荧光定量PCR检测。结果常规荧光定量PCR检测检出分枝杆菌75例(60%)阳性,其中结核杆菌74例(59.2%),非结核分枝杆菌1例(0.8%),阳性病例平均阳性基因拷贝数6.35×105/ml/例;EDTA修复后荧光定量PCR检测检出分枝杆菌88例(70.4%),其中结核杆菌83例(66.4%),非结核分枝杆菌5例(4%),阳性病例平均阳性基因拷贝数7.36×106/ml/例。两组间差异均有统计学意义(P0.05,P0.01)。结论 EDTA热修复后荧光定量PCR检测可以大幅度提高炎性肉芽肿组织石蜡切片的结核/非结核分枝杆菌的阳性检出率。     

冷冻切片后甲醛固定石蜡包埋组织DNA质量分析

目的分析冷冻切片后再行甲醛固定、石蜡包埋组织的DNA质量,探讨其在分子病理检测中的应用价值。方法收集已行术中快速冷冻病理诊断的肺腺癌14例,分别提取同一病例冷冻切片后剩余组织蜡块及常规包埋组织蜡块DNA,检测DNA浓度及OD260/OD280值,行内参基因PCR扩增以检测DNA完整性,并以ARMS法行EGFR基因突变检测。结果冷冻切片后固定包埋组织提取DNA浓度及OD260/OD280均值,分别为157.33 ng/μL及1.96,均较常规包埋组织低;所有冷冻后包埋组织提取的DNA均具有100 bp及200 bp的PCR产物,但仅部分能扩增出300 bp及400 bp的PCR产物,DNA完整性稍逊于常规固定包埋组织;冷冻后包埋组织与常规包埋组织EGFR基因突变检测结果完全一致。结论组织冷冻后再行甲醛固定及石蜡包埋,会使得组织DNA进一步降解及片段化,但仍适用于ARMS法的分子病理学检测。     

改良痰结核分枝杆菌L型涂片检测方法的建立和应用

目的建立一种快速简便、灵敏度高、特异性好的痰结核分枝杆菌L型的涂片染色检测方法。方法对常规的抗酸染色法进行方法学改良,以特异性基因扩增诊断法为标准,分析评价改良方法的灵敏度、特异性和总有效检出率等统计学指标;分别采用本改良抗酸染色法（以下简称本改良法）、常规抗酸染色法和改良IK抗酸染色法检测468例临床标本,对三种方法的阳性检出率进行统计学比较。结果本改良法阳性检出率与特异性基因扩增诊断法、改良IK抗酸染色法比较均无明显差异（χ2=0.25,P=0.6171;U=0.155,P=0.4404）,但明显高于常规抗酸染色法（U=3.713,P〈0.0010）;本改良法灵敏度、特异性和总有效检出率与改良IK抗酸染色法相似,但高于常规抗酸染色法。同时,本改良法所需时间少于改良IK抗酸染色法（5minvs24h）。结论成功建立了一种快速简便、灵敏度高、特异性好的痰结核分支杆菌L型的涂片染色检测方法,适合临床推广应用。     

聚合酶链反应检测结核分枝杆菌DNA诊断关节结核的临床评价

目的评价聚合酶链反应(polymerase chain reaction,PCR)检测关节结核标本结核分枝杆菌脱氧核糖核酸(deoxyribonucleic acid,DNA)诊断关节结核的临床价值。方法对20份标准标本(5份结核分枝杆菌标准菌株、5份卡介苗和10份其它细菌标本)分别应用PCR盲法检测结核分枝杆菌DNA。对95例关节结核标本和98例非关节结核标本分别应用PCR检测结核分枝杆菌DNA。应用疾病诊断方法的临床评价原则对PCR诊断关节结核的敏感性、特异性和准确性进行评价。结果 (1)20份标准标本PCR检测中,结核分枝杆菌和卡介苗均为阳性,而其它细菌均为阴性;(2)95例关节结核标本中,PCR阳性78例、阴性17例;98例非关节结核标本中,PCR阳性9例、阴性89例;(3)PCR检测关节结核标本结核分枝杆菌DNA的敏感性为82.11%、特异性为90.81%、准确性为86.60%、阳性预测值为89.80%、阴性预测值为84.00%;(4)PCR整个检测过程自动化控制,可在3~6h内完成。结论 PCR是一种敏感、特异、快速、简便、无创、标本微量的关节结核标本结核分枝杆菌检测方法,对于关节结核的早期、快速诊断与鉴别诊断具有极其重要的临床价值。     

Value of PCR amplification from formalin-fixed paraffin-embedded tissues in the diagnosis of Mycobacterium tuberculosis infection

Mycobacterium tuberculosis infection remains a major health problem throughout the world. In France, tuberculosis is endemic, particularly in the Paris area (Ile-de-France) and in the south (Provence Alpes c?te d'Azur) where immigration is greater than in other countries in northern Europe. Culture is the gold standard for diagnosis of tuberculosis and is the only method enabling a study of strain sensitivity to treatment. Histology contributes to diagnosis in most cases by revealing typical necrotizing granulomatous lesions. The diagnosis is then confirmed by the detection of acid-fast bacilli with Ziehl-Neelsen staining. However, the Ziehl-Neelsen stain is not sensitive and does not allow identification of different species. The polymerase chain reaction (PCR) DNA amplification method has been used to detect M. tuberculosis in formalin-fixed paraffin-embedded tissues. The aim of the present study was to investigate the value of this method for the diagnosis of M. tuberculosis infection. The results obtained with PCR assay were compared to those obtained with histological and microbiological methods (direct examination and culture). Sixty-three specimens (mainly lymph node and lung specimens) exhibiting a positive culture for M. tuberculosis were analyzed. Tuberculosis granulomas were noted in 32/63 cases, tuberculoid granulomas in 18/63, pyoepitheloid granuloms in 10/63, and non-specific inflammation in 3/63. Ziehl-Neelsen staining was positive in 11/63 cases. PCR assay on tissue sections was positive for M. tuberculosis in 58/63 cases. Controls of the PCR method (granulomas due to other mycobacterial species, foreign body granulomas, sarcoidosis granulomas) were all negative. This study shows that PCR from deparaffinized sections, 1) greatly increases the sensitivity of diagnosis of tuberculosis, 2) enables the diagnosis of M. tuberculosis infection. However, although this method reduces the time to diagnosis, culture remains the gold standard for identification of the mycobacterium and for determining the sensitivity of the isolated strain to treatment.     

结核分枝杆菌相关IFN-γ释放试验在结核病诊断及疗效监测中的应用

目的：目的：探讨结核分枝杆菌相关γ-干扰素释放试验（TB-IGRA）在结核病诊断及临床抗结核治疗效果评价中的应用价值。方法：对196例临床确诊结核患者进行相关病史收集，同时进行TB-IGRA、结核菌素皮肤试验、结核杆菌培养、涂片镜检、结核抗体检测，结核患者的治疗过程中按用药疗程（初治患者第2、5、6个月，复治患者第2、5、8个月）对其进行随访，在治疗结束后6个月和1年后各随访一次，比较TB-IGRA与结核菌素皮肤试验在治疗过程中的变化。结果：确诊结核病例196例，其中肺结核158例，肺外结核38例，TB-IGRA在结核诊断中灵敏度为82．1％，特异性为91．7％，阳性预测值为93．1％，阴性预测值为79．3％，与传统实验室检查方法相比有显著性差异（P〈0．01）。在结核患者的抗结核治疗过程中，TB-IGRA阳性率及浓度水平治疗前与治疗后2、6月以及复治后2、5、8月比较显著下降（P〈0．01），TB—IGRA水平与结核分枝杆菌数量存在较好相关性，与传统的TST方法评估疗效相比有显著性差异（P〈0．01）。结论：TB-IGRA在结核诊断中敏感性和阳性预测值较高，可作为较好的结核诊断的依据，同时TB-IGRA对肺外结核的灵敏度较高，为肺外结核患者的诊断提供了较为可靠的依据，通过治疗过程TB—IGRA检测分析，外周血TB-IGRA的变化随着抗结核治疗的进程下降的趋势明显，与结核涂片镜检结果符合率较高，是临床疗效评估较为理想的指标。     

Rapid detection of Mycobacterium tuberculosis in various clinical specimens by using polymerase chain reaction combined with a nonradioactive hybridization system

A DNA amplification system using the polymerase chain reaction (PCR) combined with a nonradioactive digoxigenin-labeled probe hybridization was employed to detect Mycobacterium tuberculosis in clinical specimens. One hundred and thirty specimens were tested by several methods including routine culture method, acid-fast staining, BACTEC 460 detection system, PCR, and PCR-hybridization techniques. Sixteen out of 130 specimens were culture positive on Middlebrook 7H11 agar, 10 were positive with acid-fast staining, 18 were positive with BACTEC 460 detection system, 23 were positive with PCR technique, and 62 were positive with PCR-nonradioactive hybridization technique. When compared with culture results, PCR-nonradioactive hybridization had an overall sensitivity of 100% (16/16) and a specificity of 59.7% (68/114). However, 28 out of 46 (60.9%) PCR-nonradioactive hybridization positive specimens which were culture negative had clinical data supporting the diagnosis of tuberculosis. In addition, 4 specimens which were negative by routine culture but positive by BACTEC 460 detection system and two specimens which were negative by routine culture but positive by acid-fast staining were all positive by PCR-hybridization technique. These data suggest that routine culture method may not be sensitive enough to detect M. tuberculosis in all kinds of clinical specimens. Taking this deviation into account, the specificity of PCR-nonradioactive hybridization technique may be rectified range from 63% (68/108) to 79.1% (68/86). PCR itself is not satisfactory enough to detect M. tuberculosis in specimens (the sensitivity and specificity were 56.3% and 87.7%, respectively) in this study. However, when it combines with DNA hybridization technique, they can be a very powerful and rapid diagnostic tool to detect M. tuberculosis in clinical specimens.     

实时荧光PCR与BACTEC MGIT 960快速培养在初治痰涂片阴性未受抗结核治疗的肺结核诊断中的应用

目的:探讨实时荧光PCR与BACTEC MGIT 960分枝杆菌快速培养(快速培养法)准确诊断初治痰涂片阴性(涂阴)且未经受抗结核治疗的肺结核可能性。方法:实验组:368份临床诊断活动性肺结核患者的初治涂片阴性痰标本;对照组:55份非结核性肺部疾病患者的痰液标本。用FQ-PCR技术、快速培养法及改良罗氏培养参考法对两组标本进行分析,观察指标为TB-DNA和结核菌阳性率。结果:实验组和对照标本中FQPCR技术、快速培养法和改良罗氏培养参考法检测的TB-DNA或结核菌阳性率分别为:16.85%、23.37%、22.01%和1%、0%、0%。三种方法分别平均耗时3h、9.6d和28d。以改良罗氏培养参考法为参照,FQ-PCR法和快速培养法的敏感性分别为69.1%和100%;特异性分别为91.2%和98.6%。结论:FQ-PCR和快速培养法均能提高初治涂阴肺结核患者的早期确诊率,但FQ-PCR的敏感性尚需进一步提高。     

Application of PCR from the fine needle aspirates for the diagnosis of cervical tuberculous lymphadenitis.

Tuberculosis remains a major public health problem worldwide. A definitive and accurate diagnosis of tuberculosis in cervical lymphadenopathy is important because satisfactory results can be achieved with chemotherapy alone, obviating surgery. Recently, fine needle aspiration cytology (FNAC) has provided an alternative and easy procedure for collection of material for cytomorphologic and bacteriologic examination. But the detection rate for M. tuberculosis from the aspirate material is still low with Ziehl-Neelson stain and even with culture. The authors therefore performed polymerase chain reaction (PCR) for mycobacterial DNA sequences in 31 cases of cytodiagnosis of tuberculous lymphadenitis and compared conventional bacteriologic methods. Ziehl-Neelson staining for acid-fast bacilli (AFB) was positive in 3 cases (10%) in direct smears, and the cultures for M. tuberculosis were positive in 6 cases (19%). In 19 (61%) among 31 samples, mycobacterial DNA fragments were detected, using the PCR method. With combined conventional and PCR method, the rate of detection was increased to 68 percent high. In conclusion, PCR is the most sensitive technique in the demonstration of M. tuberculosis in patient with clinically suspected as tuberculosis, who have AFB stain or culture negative cytology. Combined conventional and PCR methods as well as cytologic findings are of further help in the detection and characterization of M. tuberculosis.     

γ-干扰素释放试验在结核病诊断中的应用

目的 探讨γ-干扰素释放试验（IGRAs）在结核病诊断中的应用价值。方法 选取2017年7月~2019年6月在景德镇市第一人民医院就诊的100例疑似结核感染患者和50例健康体检者作为研究对象,分别行γ-干扰素释放试验和涂片抗酸染色镜检。将最终确诊为结核病患者48例设为结核病组,52例非结核病患者作为非结核病组,50例健康体检者设为正常对照组。分析三组TB-IGRAs阳性率,对比肺结核病组涂阴与涂阳TB-IGRAs检测结果,并比较TB-IGRAs与涂片抗酸染色对结核病的诊断效能。结果 结核病组IGRAs阳性率高于非结核病组和正常对照组,差异有统计学意义（P＜0.05）;涂阳肺结核和涂阴肺结核患者的IGRAs阳性率比较,差异无统计学意义（P＞0.05）;IGRAs法敏感度为85.42%,特异性为89.22%,阳性预测值为78.85%,阴性预测值为92.86%;涂片抗酸染色法敏感度为22.92%,特异性为100.00%,阳性预测值为100.00%,阴性预测值为73.38%; IGRAs诊断结核病的敏感度高于涂片抗酸染色,差异有统计学意义（P＜0.05）。结论 IGRAs具有较高的敏感度和阴性预测值,对结核病的诊断具有较高的诊断价值,值得在临床中应用。