Novel PCR-EIA method for the detection of Chlamydia pneumoniae in respiratory specimens

We report the development of a microtitre plate-based PCR-EIA assay (ELAHA; Enzyme Linked Amplification and Hybridization Assay) for the sensitive and specific detection of Chlamydia pneumoniae in sputum samples from patients with chronic obstructive airways disease (COAD). Following PCR amplification of a segment of the chlamydial heat shock 60 protein gene, the 587 bp sized amplicon is captured onto the streptavidin coated surface of a microtitre plate using a C. pneumoniae specific biotinylated probe and the level of captured product is subsequently determined via a colorimetric reaction using an automated plate reader. The ELAHA is a simple, rapid and inexpensive method for detection of low levels of infectious agents and is readily adaptable to current clinical laboratory equipment. The assay was evaluated with a cohort of hospital respiratory patients: (i) COAD patients with acute exacerbation, (ii) COAD patients without exacerbation (stable) and (iii) a non-respiratory control group. The ELAHA produced 6/12 (50%) C. pneumoniae positives in the COAD with exacerbation group, 3/13 (23%) positives in the COAD without exacerbation group and only 1/6 (17%) positives in the control non-respiratory group. This sensitive and robust PCR-EIA method can provide clinically relevant diagnostic evidence of current C. pneumoniae infection contributing to serious respiratory tract diseases such as COAD.