Failure to detect Chlamydia pneumoniae in senile calcific aortic stenosis or calcified congenital bicuspid aortic valve by immunofluorescence, polymerase chain reaction and electron microscopy

Introduction: Chlamydia pneumoniae has been identified in arterial atherosclerosis. Aortic valves affected by senile calcific aortic stenosis (SCAS) or calcification of a congenital bicuspid valve (CCBAV) may have interior environments similar to atherosclerosis. This study aimed to detect C. pneumoniae within either SCAS or CCBAV. Methods: 60 valves showing either SCAS (n=36) or CCABV (n=22) and control valves (n=2) were studied for the presence of C. pneumoniae by the following three techniques: (1) indirect immunofluorescence (IF) was performed on 36 SCAS valves, 22 CCBAV valves and 2 control aortic valves using a HEp-2 cell line infected with C. pneumoniae as a positive control. Negative controls comprised duplicate slides of the same valves with omission of the primary antibody step. A section of human stomach was also used as a negative control. A semiquantitative scoring method was used to grade positive IF staining. (2) Polymerase chain reaction (PCR) was performed on 30 SCAS valves, 20 CCBAV valves and 1 control valve using C. pneumoniae as a positive control and negative controls comprised a Ureaplasma sp. and human DNA from peripheral blood mononuclear cells. (3) Electron microscopy (EM) was performed upon 13 SCAS, 8 CCBAV and 2 control valves. Results: All three methods failed to detect the presence of C. pneumoniae in any of the 60 aortic valves examined. False positive IF staining was encountered in 81% of test valves and in 76% of negative control valve sections (positive in calcified material due to nonspecific binding of FITC-conjugated secondary antibody). No staining was observed in the negative control stomach sections. Conclusions: This study failed to detect C. pneumoniae within aortic valves showing SCAS or CCBAV. Studies using IF alone to detect C. pneumoniae in calcified tissues should be interpreted with caution, since nonspecific binding of FITC-conjugated secondary antibody by calcium in the cusps may be misinterpreted as evidence of Chlamydia. The use of appropriate controls and ancillary methods for the identification of C. pneumoniae are important in this regard.