【作者】 王平；

【导师】 朱元珏；

【作者基本信息】 北京协和医学院 ， 呼吸内科， 2011， 博士

【摘要】 目的：建立临床肺活检组织结核分枝杆菌分子快速检测方法，并评价该方法敏感性、特异性。进一步应用该方法研究结节病患者肺组织结核分枝杆菌基因检出情况，评价该方法对肺结节病及肺结核鉴别诊断价值。方法：收集北京协和医院2008年至2009年经支气管镜肺活检、经皮肺穿刺活检、电视胸腔镜手术(Video assisted thoracoscopic surgery,VATS)或开胸手术肺活检组织标本，选取诊断明确临床资料完整者共110例。肺结核病组47例(抗酸染色和/或分枝杆菌培养阳性20例，组织病理学诊断21例，临床诊断6例)，对照组63例(肺结节病组23例，肺癌组10例，肺真菌组19例，肺细菌组11例)。提取组织DNA，巢式PCR(Nested polymerase chain reaction, nPCR)方法扩增结核分枝杆菌特异插入序列IS6110基因。同时收集组织细菌、真菌、分枝杆菌涂片和培养等病原学结果以及组织病理结果等临床资料。结果：IS6110-nPCR检测下限为100fg DNA模板量(约20条结核分枝杆菌)。36例(76.6%)肺结核组织扩增阳性，52例(82.5%)非结核肺组织扩增阴性。肺结核组nPCR阳性者中具有肉芽肿等典型病理表现比例高于nPCR阴性者，而组织抗酸染色阳性、分枝杆菌培养阳性、经皮肺穿刺/TBLB组织、VATS/开胸肺组织、新鲜组织、FFPE组织比例无显著性差异。11例(23.4%)肺结核组扩增阴性，11例(17.5%)对照组扩增阳性，其中肺结节病组6例，肺真菌组5例。26.1%肺结节病患者肺组织可检出结核分枝杆菌DNA,极显著低于肺结核组(P<0.001)。结论：nPCR扩增IS6110基因检测肺组织标本诊断敏感性达76.6%,特异性82.5%,与组织病理诊断方法结合敏感性进一步提高至87.2%。组织病理具有肉芽肿等典型表现的肺组织检测敏感性更高，而肺组织抗酸染色、分枝杆菌培养等病原学结果、组织来源及组织类型均不影响检测结果。该方法诊断肺结核感染假阳性率17.5%,假阴性率23.4%,假阳性见于肺结节病和肺真菌感染患者。结核分枝杆菌与肺结节病存在相关性，但肺结节病组织结核分枝杆菌DNA检出率明显低于肺结核病组织，因此该方法可作为两者分子鉴别诊断方法目的：调查肺分枝杆菌感染菌种分布、临床特征、耐药情况及治疗结局，进一步比较在免疫抑制人群与普通人群间的差异。结合临床特征及治疗结局探讨肺非结核分枝杆菌感染危险因索、耐药菌感染危险因素及死亡等不良结局危险因索。方法：北京协和医院2009年1月至2010年7月分离自呼吸系统标本的分枝杆菌临床背景资料完整并复苏培养成功者共38株。收集其人口学资料、胸部影像学资料、基础疾病、接受免疫抑制治疗情况、抗结核治疗方案等临床资料,并随访至2010年12月收集治疗结局资料。鉴别培养基培养法菌种鉴定,比例法药物敏感性试验检测对一线抗结核药物敏感性,其中对任一种一线抗结核药耐药菌株加行二线抗结核药药敏实验。CTAB法提取菌株DNA扩增IS6110基因和rpoB基因并测序比对进行分子菌种鉴定；对鉴定为MTBC菌株的DNA PCR扩增RFP、INH、EMB、SM耐药相关基因并测序比对检测耐药突变。结果：38株分枝杆菌分离自痰标本16株,支气管吸取物或灌洗液8株,胸腔积液10株,肺组织4株。18株(47.4%)来自非HIV感染免疫抑制患者。6株(15.8%)为NTM。肺NTM感染与MTBC感染者比,平均年龄69.8岁显著增高；1/3患者有COPD、支扩等结构性肺疾病,极显著增高；胸CT上团块影表现显著增高,而胸腔积液表现显著减少,男性结节/团块影表现极显著高于女性,女性支扩表现极显著高于男性；肺外器官受累比例显著降低：治疗失败率高达40.0%,显著升高。22.2%免疫抑制患者出现脏器衰竭并发症，高于非免疫抑制患者；死亡率为43.8%,极显著高于非免疫抑制患者；两者间NTM感染率、耐药结核感染率无差异。32株MTBC中6株(18.8%)为耐药菌株,其中9.4%为单药耐药,6.3%为多药耐药,3.1%为XDR-TB。四种一线抗结核药初始耐药率依次为SM 1 8.8%、INH 9.4%、PFP和EMB各3.1%。耐药结核治疗失败率达33.3%,极显著高于敏感结核分枝杆菌感染者。35例有随访资料患者平均随访14.3月,23例(65.7%)治疗有效,4例(11.4%)治疗失败,8例(22.9%)死亡。死亡发生于结核诊断6月内,有脏器衰竭并发症者则在3月内。死亡组年龄显著大于非死亡组；多出现于胸CT上双肺多发或弥漫病变者；62.5%结核诊断时有脏器衰竭并发症,特别是呼吸衰竭、感染性休克,极显著高于非死亡组；75.0%死亡患者为免疫抑制患者，显著高于非死亡组；62.5%为治疗中断或未抗结核治疗者,极显著高于非死亡组。REP、INH、SM耐药株检测到的耐药相关基因突变模式为rpoB基因S531L点突变、katG基因S315T点突变、rpsL基因K43R突变,katGR463L点突变在INH敏感株和耐药株检出率无显著性差异。结论：临床上肺分枝杆菌感染患者15.8%为NTM感染,最常见菌种为脓肿-龟分枝杆菌群和胞内分枝杆菌。NTM感染发热少见,以咳嗽、咯血等慢性肺部疾病症状为主；影像学上团块影多见而胸腔积液少见,团块/结节影多见于男性而支扩表现多见于女性；主要累及肺部而肺外器官同时受累少见。NTM肺部感染危险因素为高龄和结构性肺基础病。非HIV感染免疫抑制患者无特异性临床症状和胸部CT表现，也不是NTM、耐药结核分枝杆菌易感危险因素,但出现脏器衰竭并发症风险升高,死亡率显著升高。其他死亡相关危险因素为高龄、双肺多发/弥漫病变、治疗中断/未治疗。rpoB、katG、rpsL基因突变与RFP、INH、SM耐药表型有较好相关性。更多还原

【Abstract】 Objective: We attempted to apply the nested PCR technique for the molecular diagnosis of Mycobacterium tuberculosis directly from clinical lung tissue samples. To evaluate the sensitivity and specificity of this method. To assess the relationship between MTBC and pulmonary sarcoidosis by examination of mycobacterial DNA in lung tissue samples of sarcoidosis,and to assess the differential diagnostic value of this method in pulmonary sarcoidosis and pulmonary tuberculosis.Method: 110 lung tissue specimens from 110 patients at Peking Union Medical Hospital from 2008 to 2009 were collected. Among them, 47 cases were diagnosed as pulmonary TB (AFB stain and/or mycobacetrial culture were positive in 20 cases, 20 cases were diagnosed by typical histopathological findings, and 6 were diagnosed clinically) , 23 were pulmonary sarcoidosis, 10 were lung cancer, 19 were pulmonary fungal infection and 11 were pulmonary bacterial infection except MTBC. DNA was extracted and MTBC DNA was tested using nested PCR, the target for the amplification being a segment of IS6110 gene. Their medical records were examined to gather the microbiogic and histopathologic data.Results:The minimum amount of DNA necessary for a positive results was 100fg, ,corresponding to 20 mycobacterial cells. nPCR was positive in 36(76.6%) of 47 patients with pulmonary TB, and nPCR was negative in 52(82.5%) of 63 patients with non-pulmonary TB. 82.6% of nPCR-positive patients in pulmonary TB group demonstrated granulomatous inflammation, higher than nPCR-negative patients in the same group. However the differences between the rate of positive AFB stain and mycobacterial culture, TBLB or percutaneous lung biopsy samples, and fresh tissue samples were not significant. nPCR was positive in 11(17.5%) of 63 patients with non-pulmonary TB. Among them 5 were from pulmonary fungal infection group and 6 were from pulmonary sarcoidosis group. The genome of MTBC can be detected in 26.1% of patients with pulmonary sarcoidosis. But the Frequency was significantly lower than that of pulmonary TB group.Conclusion:The sensitivity and specificity of this method was 76.6% and 82.5% respectively. A higher proportion of paitienls with pulmonary TB was diagnosed when nPCR was combined with histopathologic results. The frequency of positive nPCR results was higher in the pulmoary TB patients whose hi slopathol ogi c findings showed granulomatous inflammation. However the nPCR examine results has no relation to the microbiologic findings, sampling method and whether fresh tissue or FFPE tissue. False positive rate i s 17.5%, False positive was seen in the patients with pulmonary sarcoidosis or fungal infection. There was association between MTBC and some cases of pulmonary sarcoidosis. nPCR amplifing the TS6110 gene of V1TBC is a valuable molecular diagnostic method for differential ion between sarcoidosis and tuberculosis. Objective: To investigate species distribution, clinical features, drug-susceptibility, treatment outcome, and prgnostic factors of pulmonary mycobacterium infection. To identify risk factors for pulmonary NTM and drug-resistant M. tuberculosis infection. To compare the difference between non-HIV immunosuppressed patients and non-immunosuppressed patients.Method: A total of 38 mycobacterium clinical strains isolated from respiratory specimens were obtained from patients hospitalized at Peking Union Medical College Hospital from Jan 2009 to July 2010.Their medical records were examined to gather clinical, radiological,and follow-up data. 1dentification of mycobacterial species was performed through using differential culture medium and sequencing analysis of rpoB gene. Drug susceptibility testing was performed by using proportion method. DNA was extracted by CTAB method. Sequencing analysis of mutation conferring resistance to RFP, INH, SM, and EMB in rpoB gene, katG gene, inhA gene, inhA promoter, rrs gene, rpsL gene, and embB gene was performed in drug-resistant M. tuberculosis strains.Results: 16 strains were isolated from sputum, 8 from bronchoalveolar washings or tracheal aspirates, 10 from pleural fluid, and 4 from lung tissue specimens. 18 strains (47.4%) were from nor-HIV immunosuppressed patients. 6 strains (15. 8%) were identified as NTM. Compared wi th pulmonary TB patients, patients with pulmonary NTM infection had a higher average age, more structural lung disease, more masslike shadow on lung CT, less pleural effusion, and less extra-pulmonary organ involving. Masslike/nodular shadow was more frequently seen in male patients. On the contrary, bronchiectasis was more frequently seen in female patients. 40% of NTM infection cases had treatment failure. Compared with non-immunosuppressed patients, the immnunosuppressed pat ients had more complication of organ failure and higer mortalityrate. The infection rate of NTM and drug-resistant M. tuberculosis had no significant difference between the two groups. 6 strains (18.8%) of M. tuberculosis were resistant to at least one of four first-line antituberculosis drugs. Initial drug-resistant rate in descending order is SM 18.8%, INH 9.4%, RFP and EMB 3.1%, respectively. 33.3% of drug resistant patients had treatment failure, significant higher than drug sensitive patients. Average follow-up period was 14.3 months. 23(65.7%) of patients improved, 4(11.4%) failed, and 8(22.9%) died. Dead patients had a higer average age, more bilateral multiple/diffuse lesion on lung CT, more complication of organ failure espcially respi ratory failure or septic shock, more immunosuppressed cases, and higher treatment default or no treatment rate. The mutation S531L in rpoB gene, S315T in katG gene, K43R in rpsl gene were detected only in RFP, INH, and SM-resistant strains. The mutation R436L in katG gene was found in both INH-resi slant and INH-sensitive strains.Conclusion: 15.8% of patients were infected by NTM. The most common species were MCAG and M. intracellulare. Fever, pleural effusion, and extra-pulmonary involving were less common in NTM lung disease. Masslike/nodular shadow was male predominance and bronchi ectasis was female predominance. Risk factors for pulmonary NTM infection was old age and structural lung diseases. Non-HIV immnunosuppressed patients has no specific symptoms and plumonary manifestat ion, and were not risk factor for pulmonary NTM and drug-resistant M. tuberculosis infection. However, the risk for complications of organ failure and mortality elevated. Other risk factors for mortality included old age, bilateral multiple/diffuse lesions, treatment default or no treatment. There were good agreement between the mutation in rpoB gene, katG gene, rpsL gene and phenotypic resistance.更多还原