Abstract
Chlamydia are Gram-negative, intracellular pathogens colonizing the epithelial mucosa. They cause primarily atypical pneumonia and have recently been associated with chronic diseases. Diagnostics rely almost exclusively on serological methods; PCR tests are used rarely because in patients with positive ELISA, it is nearly impossible to identify chlamydial DNA. To understand this issue, we elaborated a reliable and sensitive nested PCR method (panNPCR) for identifying all Chlamydiales species, not only in sputa, but also in clotted blood. Sequencing of the PCR product revealed that 41% of positive sputa samples and 66% of positive blood samples were not infected by Chlamydia but with "Chlamydia-related bacteria" such as Rhabdochlamydia sp., Parachlamydia sp., Protochlamydia sp., Neochlamydia sp., Mesochlamydia elodeae and lacustris, Piscichlamydia salmonis, and Estrella lausannensis. Consequently, we propose that there might be more than four human pathogenic Chlamydia species. We did not find any clear correlation between increased levels of antibodies and the presence of their DNA. Chlamydialles DNA was found in sputa samples from individuals positive for IgG or IgA but not in blood samples. Thus, elevated IgG and IgA levels are not reliable markers of chronic infection, and the presence of persistent forms should be proved by panNPCR. Apparently, the differences between ELISA and DNA amplification results have three main methodological reasons. The first one is the threshold occurrence of chlamydial genetic material in sputum and blood. The second one is the fact that a significant part of the samples can have DNA with sequences different from those of other species of the order Chlamydiales. The third one is the high background characteristic for ELISA, the absence of paired sera, and the vague interpretation of the gray zone.