【作者】 武玮；

【导师】 潘毓萱； 王甦民；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 2001， 硕士

【摘要】 对结核病进行快速、敏感、特异的细菌学诊断和化疗疗效监测是临床结核病诊治中迫切需要解决的问题。核酸扩增技术快速、敏感、特异，但不能辨别被测菌的活力状态，且临床应用研究尚未成熟；而培养法能检测出有活力菌的生长，培养阳性是结核病细菌学诊断的金标准，但其检测需时长。因此，本研究应用7H12血清半流体培养和PCR扩增相结合的方法对痰涂片阴性肺结核病人痰标本进行检测，以期达到对结核菌早期检定和确认活菌的目的：同时对PCR的临床意义进行探索，尝试说明PCR在结核病诊断和化疗监测方面的临床应用价值。 本研究对早期诊断组的46份痰标本和化疗监测组的24份痰标本分别进行了涂片、培养和PCR检测，结果显示：对于涂片阴性样本，SLCP、改良罗氏和7H12半流体培养所需的平均检出时间分别为3.45天、29.56天和16天，可见，SLCP所需检出时间明显缩短(P<0.01)；敏感性方面，涂阴标本的SLCP检测阳性率0天时为62.07％，与改良罗氏培养和7H12血清半流体培养的8周时阳性率相同，而SLCP的检测阳性率于7天时上升到86.21％，较0天时有明显增长(P<0.05)。另外，7H12血清半流体培养的平均检出时间较改良罗氏固体培养明显缩短(P<0.01)，可以应用7H12血清半流体培养对PCR阳性样本进行活菌的判断。本研究应用同一样本同时进行培养和核酸扩增，结果显示，两种不同培养基分别应用、同时应用、及结合临床资料对PCR的支持率分别为76.09％、80.43％和91.11％。本文的化疗监测组结果显示，PCR检测在涂片和培养阴转后，最长至化疗7个月时仍未阴转。结论：SLCP检测方法对于肺结核的初始诊断具有一定临床应用价值，尤其对于痰涂片阴性肺结核，可以达到早期诊断和确认活菌的目的；与传统的培养法相比，PCR技术迅速、敏感、并得到培养法和临床资料的很高支持率，在标准化监控下对于肺结核的初始诊断具有一定的临床应用价值；但结核分枝杆菌DNA扩增不适用于结核病的化疗疗效监测。更多还原

【Abstract】 There’s a great need of more sensitive and specific method for the rapid diagnosis and monitoring treatment response of tuberculosis nowadays. It has proved that nucleic acid amplification techniques are rapid, sensitive and specific, but the inability of the PCR methods to detect a difference between viable and nonviable organisms limited its widespread use in clinical specimens. At present, it is considered that molecular methods have not been able to replace cultivation for the detection of mycobacteria from clinical specimens. So we developed a new method, named Middlebrook 7H12 semi-liquid culture PCR(SLCP), which combine cuture with Middlebrook 7H12 semi-liquid media and PCR to detect smear-negative sputum from patients with pulmonary tuberculosis, in order to achieve rapid diagnosis and determine viable organisms. At the same time, via this study, we try to show the clinical significance of PCR in initial diagnosis and monitoring treatment response of tuberculosis.We detected 46 specimens for rapid diagnosis and 24 specimens for monitoring treatment response of tuberculosis. We found that for smear-negative specimens, the mean time to detection of M. tuberculosis were 29.56 days in L-J media, 16 days in 7H12 semi-liquid media and 3.45 days with SLCP. As for sensitivity, at the 0 day and the 7th day, the sensitivity for SLCP is 62.07% and 86.21%, respectively. The latter is higher(P<0.05). Compared to SLCP, the sensitivity for L-J media and 7H12 semi-liquid media is 62.07% at the 8th week. We also found that the mean time to detection of M. tuberculosis in 7H12 semi-liquid media is fewer than in L-J media(P<0.01). Therefore we can use 7H12 semi-liquid media to determine viable organisms. Moreover, the support rate of L-J media or/and 7H12 semi-liquid media, and combine with clinical data is 76.09%, 80.43% and 91.11%, respectively. Besides, the results of specimens for monitoring treatment response of tuberculosis showed that the persistence of M.tuberculosis DNA in sputum as long as 7 months after the start of treatment, at the same time the specimens were smear and culture negative. From all results above, we concluded that SLCP might be useful in the initial diagnosis of tuberculosis to a certain extent, especially for smear-negative pulmonary tuberculosis. With this method, we might achieve rapid diagnosis and determine viable organisms; Under the conditions of standardization and strict quality control, PCR methods are rapid, sensitive and specific, and supported by cuture and clinical data at high rate. They could be useful in the initial diagnosis of tuberculosis, but they might be inappropriate for monitoring the response of patients to antituberculosis therapy.更多还原