肺结核患者结核菌L型感染及其荧光定量PCR检测的意义

Detection of tubercle bacilli L-forms in patients with pulmonary tuberculosis and by fluorescence quantitive PCR for clinical diagnosis

目的评价荧光定量PCR(FQ-PCR)和结核菌(MTB)L型检测的临床价值。方法收集61例痰阴性肺结核患者痰、血标本分别进行分枝杆菌及其L型培养,并采用染色、免疫组化和FQ-PCR技术进行检测和鉴定,部分标本透射电镜观察。结果痰MTB及其L型检测的阳性率为72.4%,血液阳性率为28.3%,而痰血检测总的阳性率为75.4%。痰直接FQ-PCR33.3%,培养后FQ-PCR40.4%,总阳性率45.5%。血直接FQ-PCR0/35,培养后FQ-PCR 22.5%。痰血FQ-PCR阳性率比较,χ2=5.3028,P<0.05,差异有统计学意义,痰阳性率明显高于血。痰和血FQ-PCR阳性率低于相应的涂片和培养结果。结论分枝杆菌L型的分离和FQ-PCR的应用可提高痰菌阴性肺结核的诊断效率。FQ-PCR阴性不能完全排除结核病,但可用于痰及血中分枝杆菌L型的辅助诊断,尤其对不易从感染部位取得标本的患者更有帮助。

Objective To determine the diagnostic efficacy of sputum and peripheral blood-based fluorescence quantitive polymerase chain reaction （FQ-PCR）for diagnosis of pulmonary tuberculosis.Methods Sputum and blood samples were collected from 61 patients of sputum negative pulmonary tuberculosis, and processed for mycobacteria and their L-forms by culture, ZN staining, immunohistoehemistry stain（IHC） and FQ-PCR. Part of them were re-confirmed by transmission electron microscopy（TEM）. Results Out of 61 cases,75.4% were examination positive for bacillary or L-forms of the tuberculosis agent.They are much less common in blood than in sputum;the rates were 28.3% and 72.4%, respectively. The total count of mycobacteria and their L-forms yielding positive amplification with M. tuberculosis complex-specific primers was 52.5%, with 45.5% and 22.5% in sputum and in blood（ P 〈 0.05） .The result of FQ-PCR was not consistent with smear and cultivation of mycobacteria and their L-forms. Conclusion The isolation of mycobacterial L-forms and the use of polymerase chain reaction significantly enhance the efficiency of the etiological diagnosis of spu- tum negative pulmonary tubemulosis. Due to low sensitivity, a negative PCR assay does not rule the disease. However, this test may be helpful in cases where specimens from the site of infection are not available.