Meijer A, Roholl P J, Gielis-Proper S K, Meulenberg Y F, Ossewaarde J M
Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands.
J Clin Pathol. 2000 Dec;53(12):904-10. doi: 10.1136/jcp.53.12.904.
There is a considerable discrepancy between data from the detection of Chlamydia pneumoniae in atherosclerotic lesions obtained by means of immunocytochemistry and data obtained using the polymerase chain reaction. This study evaluated methods for the in situ detection and assessment of the viability of C pneumoniae bacteria.
Chlamydia pneumoniae membrane protein, heat shock protein 60, and lipopolysaccharide were detected by immunocytochemistry, and genomic DNA and 16S rRNA by in situ hybridisation in paraffin wax embedded sections of cultured HEp2 cells infected with C pneumoniae and of lungs from mice infected intranasally with C pneumoniae.
Inclusions reactive for all three antigens, DNA, and 16S rRNA were seen in infected HEp2 cells, in all positive bronchus and alveolar epithelial cells, and in some of the positive infiltrate cells in the lungs of mice up to seven days after infection. In all alveolar macrophages and in the infiltrate cells positive for antigens only, the staining pattern was granularly dispersed throughout the cytoplasm up to seven days after infection. At 21 days after infection, only this granular staining pattern was seen for antigens in infiltrate cells and macrophages in the alveoli and bronchus associated lymphoid tissue. At this time point, DNA or 16S rRNA were detected sporadically, but always as inclusion-like staining.
Because antigens with an inclusion-like staining were detected only together with DNA and 16S rRNA, this type of staining pattern suggested the presence of viable bacteria. Thus, the granular staining pattern of antigens in the absence of staining for DNA and 16S is most likely caused by non-viable bacteria. In conclusion, these methods are suitable for the in situ detection of C pneumoniae and the assessment of its viability.
通过免疫细胞化学方法检测动脉粥样硬化病变中肺炎衣原体所获得的数据，与使用聚合酶链反应获得的数据之间存在相当大的差异。本研究评估了肺炎衣原体细菌原位检测及活力评估的方法。
通过免疫细胞化学检测肺炎衣原体膜蛋白、热休克蛋白60和脂多糖，通过原位杂交检测培养的感染肺炎衣原体的HEp2细胞石蜡包埋切片以及经鼻感染肺炎衣原体的小鼠肺组织石蜡包埋切片中的基因组DNA和16S rRNA。
在感染的HEp2细胞、所有阳性支气管和肺泡上皮细胞以及感染后长达7天的小鼠肺组织中部分阳性浸润细胞中，可见对所有三种抗原、DNA和16S rRNA呈反应性的包涵体。在所有肺泡巨噬细胞以及仅对抗原呈阳性的浸润细胞中，感染后长达7天，染色模式呈颗粒状分散于整个细胞质中。感染后21天，在肺泡和支气管相关淋巴组织的浸润细胞和巨噬细胞中，仅见抗原呈这种颗粒状染色模式。此时，DNA或16S rRNA偶尔被检测到，但总是呈包涵体样染色。
由于仅在同时检测到DNA和16S rRNA时才检测到呈包涵体样染色的抗原，这种染色模式提示存在活细菌。因此，在无DNA和16S染色时抗原的颗粒状染色模式很可能是由非活细菌引起的。总之，这些方法适用于肺炎衣原体的原位检测及其活力评估。
