Laboratory diagnosis of C. pneumoniae infections can be performed by culture, serology, and nucleic acid amplification tests. In tissues, chlamydiae can additionally be targeted by immunohistochemistry and in situ hybridization techniques.
The most frequently used approach for the diagnosis of C. pneumoniae infections is serology. Although, often, it can only retrospectively demonstrate a significant rise in antibody titers. In primary infection, specific IgM is detectable from 2 to 3 weeks until 2 to 6 months after infection. IgG antibodies may not appear until 2 months after infection. In reinfection, complement fixation as well as IgM antibodies often remain negative while the IgG titer may rise to 1:512 or more as soon as 1 week after infection.[28,29] There are yet no validated serologic markers for persistent or chronic infection, but IgA antibodies in combination with high titers of IgG antibodies may be indicative.
The most used and recommended serologic test is the microimmunofluorescence test (MIF), which uses purified formalinized EBs from C. pneumoniae, C. trachomatis, and C. psittaci that are fixed on glass slides in distinct, small spots. It can therefore simultaneously determine species-specific antibodies to the three chlamydiae. The chlamydial complement fixation test, based on the common lipopolysacharide antigen (LPS), may react more promptly, but has a low sensitivity for reinfection. It does not distinguish between various species of chlamydiae and is therefore generally not recommended. The same is valid for whole inclusion fluorescence tests that are not species-specific either and not yet sufficiently validated to be recommended. Enzyme immunoassays (EIA) offer a more automated and standardized workflow. Evaluations of these tests are currently underway.
Culture of C. pneumoniae is hampered by great technical effort and limited sensitivity. Therefore it is not used routinely, but remains essential for the determination of resistance and the characterization of strains for research purposes. Cultured chlamydiae can be identified by PCR or by species-specific antibodies (Fig. 1). We have recently developed a probe set for differentiation of chlamydiae by fluorescent in situ hybridization (FISH) that can be implemented for this purpose and may also be used for the detection of chlamydiae in clinical samples (Fig. 1).
A variety of DNA amplification methods has been described using different formats (nested, nonnested, real-time LightCycler PCR, and enzyme time-released touch down PCR.) and targeting different gene sequences including the ompA gene, 16SrRNA gene, omcB, and the Pst I fragment. These methods are increasingly used to detect C. pneumoniae in respiratory specimens as well as in tissues; however, standardization of these methods is urgently required to avoid discrepant results.
Immunohistochemistry (IHC) is used for the detection of C. pneumoniae in tissues for research issues such as the association of chlamydiae with arteriosclerosis. Contrary to PCR and culture, this technique allows for the preservation of the tissue structure and localization of the antigen. Results, however, have to be interpreted carefully as artifacts and positive stainings are difficult to distinguish.
An additional technique preserving the histology of the tissue is in situ hybridization with labeled DNA probes.[27,31] This technique is hampered by similar problems as seen in IHC.
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