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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Meijer
A
A
Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands. Adam.Meijer@rivm.nl
Roholl
P J
PJ
Gielis-Proper
S K
SK
Meulenberg
Y F
YF
Ossewaarde
J M
JM
D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis; D018929:Cell Culture Techniques; D023521:Chlamydophila Infections / Q000175:diagnosis*; D016993:Chlamydophila pneumoniae / Q000276:immunology / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D005260:Female; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D008168:Lung / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016612:Paraffin Embedding; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S