实时荧光定量PCR在结节病与不典型结核鉴别诊断中的临床应用

Clinical application of Real-Time quantitative PCR in the differential diagnosis of sarcoidosis and atypical tuberculosis

目的验证和评价实时荧光定量PCR（polymerase chain reaction）的方法检测结核分枝杆菌DNA在结节病与不典型结核病鉴别诊断中的实际应用价值。方法以2008年6月至2009年6月期间,经病理活检证实为肉芽肿性疾病,但无法确定为结节病或结核病的49例患者为研究对象。运用我科前期建立的实时荧光定量PCR的方法检测上述患者石蜡包埋病理组织标本中的结核分枝杆菌DNA（Tuberculosis polymerase chain reaction,TBPCR）,以1.14×10~3拷贝/ml作为鉴别结节病与不典型结核病的最佳cutoff值,结合临床、影像、病理和其他检查结果帮助临床鉴别诊断并指导用药。随访所有患者至2009年8月1日,通过临床表现、影像学、治疗效果等变化来验证和评价本方法的实际应用价值。结果 49例患者定量TB-PCR结果中,10例患者结核分枝杆菌DNA为阳性（2.01×10~3～7.98×10~4拷贝/ml）（阳性率20.41%）,39例为阴性。结合患者临床资料及TB-PCR结果确定诊断,33例为结节病,2例结核病,诊断明确率达到72%（35/49）。诊断明确患者治疗后随访（2～14个月）,33例结节病和2例结核病患者经相应治疗均达到稳定或不同程度好转,无1例出现病情恶化。结论实时定量PCR法检测石蜡包埋组织中结核菌DNA能灵敏而高效地检出不典型结核病肉芽肿中的结核杆菌DNA,可作为鉴别结节病和不典型结核病的有效方法之一。

Objective To verify and evaluate the clinical application value of pre-established Real-Time quantitative PCR to detect Mycobacterium tuberculosis DNA in the differential diagnosis of sarcoidosis and atypical tuberculosis.Method Forty-nine patients with granulomatosis disciformis but no final diagnosis as sarcoidosis or tuberculosis were enrolled into this study from Jun 2008 to Jun 2009.Real-Time quantitative PCR pre-established were used to detect Mycobacterium tuberculosis DNA（TB-PCR）in 49 patients＇ paraffin blocks with the optimal cut-off value 1.14×10~3 copies/ml. Clinical,imaging,pathology and other data were combined to make differential diagnosis and give guidance of therapy.The clinical appearance,imaging changes,therapeutic effects were followed up till Aug 1st,2009 and the clinical application value of this method were evaluated.Result Ten samples of TB-PCR were positive（2.01×10~3 -7.98×10~4 copy/ml）,the positive rate was 20.41%,39 samples were negative. Combined with clinical data and TB-PCR results,33 cases were diagnosed as sarcoidosis,2 were tuberculosis. The percentage of definite diagnosis achieved was 72%（35/49）.Patients with definite diagnosis were followed up for 2～14 months in which 33 patients with sarcoidosis and 2 cases with tuberculosis got stable or improved after the therapy,none of them worsen.Conclusion The Real-Time quantitative PCR for detection of Mycobacterium tuberculosis DNA in paraffin-embedded tissues can sensitively and effectively detect Mycobacterium tuberculosis from tuberculosis proliferative granulomatous lesions.It is effective to help to differentiat sarcoidosis and atypical tuberculosis.