Evaluation of false-positive reaction with ELISA for the detection of Chlamydophila pneumoniae-specific IgM antibody in adults.

Chlamydophila pneumoniae, an obligate intracellular human pathogen, has been proven to cause respiratory tract infections (1). Among the diagnostic methods for detecting C. pneumoniae, serological testing is currently the most common tool used to make the C. pneumoniae infection diagnosis. In Asia, most clinicians and researchers use a commercial serologic test kit, enzyme-linked immunosorbent assay (ELISA) (Hitazyme C. pneumoniae Ab; Hitachi Chemical Co., Tsukuba, Japan) to detect anti-C. pneumoniae-specific immunoglobulin M (IgM), IgA, and IgG antibodies (2). In recent years, with the accumulation of data, it has been demonstrated that false-positive ELISA IgM reactivity is frequent among asymptomatic subjects, as well as in patients with acute respiratory tract infections (ARTIs) (3). Thus, this ELISA was updated with a modified cut-off value (index ≥ 1.6 to ≥2.0) and the inclusion of new absorbing reagent (anti-human IgG antibodies) to reduce the false-positive reaction (2). Using this new cut-off value and new absorbent, however, a prospective multicenter study demonstrated the low positiveconcordance rate comparing the microimmunofluorescence (MIF) test, which is the current gold standard for serologic testing of C. pneumoniae infections worldwide (1). In the present study, to clarify the false-positive reaction of a newly modified ELISA, findings were compared with those on several established laboratory tests such as culture, immunoblotting, the AniLab C. pneumoniae enzyme immunoassay (EIA; AniLab Systems Ltd., Oy, Vantaa, Finland), and the MIF test. Two groups were enrolled in this study; 60 patients with non-C. pneumoniae pneumonia (bacterial pneumonia), and 120 adult volunteers without ARTIs. Sixty adult patients with pneumonia (36 males and 24 females with a mean age of 67.9 years) were seen at Kawasaki Medical School Hospital between January 2008 and March 2009. The diagnosis of pneumonia was made by typical clinical, laboratory, and radiographic findings. These patients were diagnosed with bacterial pneumonia by culture and/or urinary antigen test methods according to standard diagnostic criteria. One hundred twenty adult volunteers without ARTIs (74 males and 46 females with a mean age of 67.1 years) who were regularly visiting the outpatient clinic during the study period were also enrolled. The subjects were excluded from the study if they reported a history of infection with C. pneumoniae, if they had taken antibiotics within the preceding 2 weeks before enrollment, or if they reported having had a clinical syndrome compatible with pharyngitis, sinusitis, bronchitis, or pneumonia within the 6 months preceding enrollment. Nasopharyngeal swab specimens and paired sera were obtained from all subjects with informed consent; the study protocol was approved by the Ethics Committee at Kawasaki Medical School. The culture, AniLab-EIA, ELISA, MIF test and immunoblotting analysis were performed as reported previously (3,4). To confirm the positive reactivity of each serological test, positive sera in the AniLab-EIA, ELISA, MIF test, and/or immunoblotting were also analyzed by another established serological test, a recombinant enzyme immunoassay based on recombinant LPS of Chlamydia (rEIA; Medac, Wedel, Germany) (3). In the preliminary study, we confirmed the positive reactions in all serological tests using the C. pneumoniae IgMpositive sera (4). C. pneumoniae IgM-positive cases as measured by the ELISA were observed in seven (11.6%) patients and 12 (10%) subjects without ARTIs (Table 1). However, cell culturing, AniLab-EIA, MIF test, and immunoblotting analysis did not reveal any positive cases in the two groups. No cases with significant increases in IgG or IgA antibody titer in the ELISA, AniLab-EIA and MIF tests between paired sera were observed. The 19 cases determined to be IgMpositive only in the ELISA were all negative by rEIA, and these positive results in the ELISA were considered to be false-positive reactions.