Chlamydia pneumoniae in vitro and in vivo: a critical evaluation of in situ detection methods

Aims-There is a considerable discrepancy between data from the detection of Chlamydia pneumoniae in atherosclerotic lesions obtained by means of immun ocytochemistry and data obtained using the polymerase chain reaction. This study evaluated methods for the in situ detection and assessment of the via bility of C pneumoniae bacteria. Methods-Chlamydia pneumoniae membrane protein, heat shock protein 60, and l ipopolysaccharide were detected by immunocytochemistry, and genomic DNA and 16S rRNA by in situ hybridisation in paraffin wax embedded sections of cul tured HEp2 cells infected with C pneumoniae and of lungs from mice infected intranasally with C pneumoniae. Results-Inclusions reactive for all three antigens, DNA, and 16S rRNA were seen in infected HEp2 cells, in all positive bronchus and alveolar epitheli al 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 i nfiltrate cells positive for antigens only, the staining pattern was granul arly dispersed throughout the cytoplasm up to seven days after infection. A t 21 days after infection, only this granular staining pattern was seen for antigens in infiltrate cells and macrophages in the alveoli and bronchus a ssociated lymphoid tissue. At this time point, DNA or 16S rRNA were detecte d sporadically, but always as inclusion-like staining. Conclusions-Because antigens with an inclusion-like staining were detected only together with DNA and 16S rRNA, this type of staining pattern suggeste d the presence of viable bacteria. Thus, the granular staining pattern of a ntigens 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.