呼吸道疾病发作期患者肺孢子菌寄植状况与肺疾病相关性研究

Relationship between Pneumocystis colonization and exacerbation of respiratory disease

目的调查呼吸系统疾病急性发作期患者肺孢子菌定植或感染状况,探讨肺孢子菌寄植与呼吸系统疾病的相关性。方法构建检测肺孢子菌线粒体大亚基核糖体核糖核酸基因的mtLSU巢式PCR反应体系,鉴定其敏感性和特异性。利用新建立的巢式PCR检测呼吸系统疾病急性发作期患者痰液中肺孢子菌基因,分析肺孢子菌在呼吸系统疾病患者中的定植率及其与呼吸系统疾病的相关性。结果新建立的mtLSU巢式PCR扩增的肺孢子菌基因序列与GenBank中人源肺孢子菌基因序列同源性为100%,且与其他8种呼吸道病原体无交叉反应。敏感性检验结果表明,扩增基因的最小量为366 fg。对98例呼吸疾病急性发作期患者的103份标本检测的结果显示,肺孢子菌基因检出率为62.14%(64/103)。结论本研究建立的mtLSU巢式PCR方法具有较高的敏感性和特异性,适用于无创性呼吸道标本肺孢子菌基因检测;肺孢子菌在呼吸系统疾病患者中有较高的定植和感染率。

Objective To investigate the colonization or infection of Pneumocystis jirovecii in patients in exacerba- tion of respiratory diseases and explore the relevance of P. jirovecii colonization to respiratory diseases. Methods A nested PCR system targeting large subunit ribosomal RNA genes of P. jirovecii mitoehondria was built and its sensitivity and specificity were identified. The mtLSU nested PCR method was performed to detect P. jirovecii in induced sputum samples from patients in exacerbation of respiratory diseases. And the P. jirovecii colonization rate in patients in respiratory diseases was analyzed. Results The sequenced DNA fragment which was amplified by newly created mtLSU nested PCR shared 100% homology with the P. jirovecii mtLSU rRNA gene announced in Gen- Bank. There was no cross-reaction with the other 8 kinds of respiratory pathogens. The sensitivity test result showed that the minimum amount of amplified gene was 366 fg. The optimized nested PCR was applied on all of the 103 clinical specimens included in the study, resulting in a detection rate of 62.14% （64/103） for the genome of P. jirovecii. Conclusion The mtLSU nested PCR established in the study havs high sensitivity and specificity. It is suitable for the detection of P. jirovecii genes in non-invasive respiratory specimens. There is a high rate of P. jirovecii colonization or infection in patients with respiratory diseases.