This could have been due to antigenically similar epitopes, but we could not exclude the possibility of co-infection as these serum samples were found to be positive by the rP1-C assay, as well (data not shown). These data suggest a low risk of cross-reactivity of this assay with an immune response to other respiratory tract bacterial infections, but more RTI serum samples should be tested to confirm these results. The false-positive results could be also explained

by cross-reactivity Vactosertib molecular weight between the rAtpD proteins of M. pneumoniae and M. genitalium, a phylogenetically closely related species to M. pneumoniae. However, we were not able to collect and study some serum samples from M. genitalium-infected patients as the diagnosis of these infections is only based on molecular methods. It would be very interesting to further include some serum samples from M. genitalium-infected patient in the study. Conclusion In summary, this study presents a new antigen, AtpD, that could contribute to improvements in the diagnosis of M. pneumoniae infection in the early and acute phase and could be more specific than the commercial assays using complex extracts. We have shown that the combination

of rAtpD with rP1-C antigen to detect IgM contributed to improvements in the early specific diagnosis selleck chemical of M. pneumoniae infection. Indeed, several studies have recently reported that combination of selected antigens provide higher sensitivity than single antigens [38]. Methods Organisms and ZD1839 nmr growth conditions The M. pneumoniae reference strain M129 (ATCC 29342) was cultured in SP4 medium containing Cell press phenol red used as pH indicator. Tissue culture flasks (Nunc) were incubated at 37°C and inspected daily for colour changes. The exponential growth phase was indicated by a colour change in medium from red to orange-yellow. The cells were harvested at this stage and washed in phosphate

buffered saline (PBS) and the pellet was stored at -20°C. Patients and healthy blood donors From January 2004 to December 2006, serum samples were retrospectively selected from 103 patients (54 children, 1-15 years of age and 49 adults, 17-82 years of age), admitted to Pellegrin hospital (Bordeaux, France), Cochin hospital (Paris, France), and Raymond Poincaré hospital (Garches, France) with a diagnosis of M. pneumoniae RTI. All of the serum samples were found to be positive for M. pneumoniae by serodiagnosis, along with a positive direct diagnosis for some patients, with either culture or PCR. Depending on the laboratory where the M. pneumoniae serodiagnosis was done, the serological methods used were either the CFT (Virion Antigen) and a commercial IgM ELISA test (Platelia EIA, Bio-Rad, ImmunoCard Mycoplasma Test, Meridian) or a combination of IgM and IgG ELISAs (ImmunoWell IgM, IgG EIA, BMD). Six paired serum samples were collected with an acute-phase sample and a convalescent sample obtained at least two weeks after the first sample.