ABSTRACT
Serologic methods are well established for the diagnosis of Mycoplasma pneumoniae infections in humans, but they are less sensitive than polymerase chain reaction (PCR). To improve their sensitivity, a new panel of antigens was tested. Compared with PCR results, up to 92% of PCR-positive patients were confirmed by our immunoblotting approach having a specificity between 92.6% and 100%.
Subject(s)
Antigens, Bacterial/chemistry , Immunoblotting/methods , Mycoplasma pneumoniae/isolation & purification , Pneumonia, Mycoplasma/diagnosis , Serologic Tests/methods , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Bacterial/blood , Child , Child, Preschool , Community-Acquired Infections/diagnosis , Community-Acquired Infections/immunology , Community-Acquired Infections/microbiology , DNA, Bacterial/analysis , Female , Humans , Infant , Male , Middle Aged , Mycoplasma pneumoniae/immunology , Pneumonia, Mycoplasma/immunology , Pneumonia, Mycoplasma/microbiology , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Reproducibility of Results , Respiratory System/microbiology , Sensitivity and SpecificityABSTRACT
BACKGROUND: Mycoplasma pneumoniae is responsible for acute respiratory tract infections (RTIs) common in children and young adults. As M. pneumoniae is innately resistant to beta-lactams antibiotics usually given as the first-line treatment for RTIs, specific and early diagnosis is important in order to select the right treatment. Serology is the most used diagnostic method for M. pneumoniae infections. RESULTS: In this study, we identified the M. pneumoniae ATP synthase beta subunit (AtpD) by serologic proteome analysis and evaluated its usefulness in the development of a serological assay. We successfully expressed and purified recombinant AtpD (rAtpD) protein, which was recognised by serum samples from M. pneumoniae-infected patient in immunoblots. The performance of the recombinant protein rAtpD was studied using a panel of serum samples from 103 infected patients and 86 healthy blood donors in an in-house IgM, IgA and IgG enzyme-linked immunosorbent assay (ELISA). The results of this assay were then compared with those of an in-house ELISA with a recombinant C-terminal fragment of the P1 adhesin (rP1-C) and of the commercial Ani Labsystems ELISA kit using an adhesin P1-enriched whole-cell extract. Performances of the rAtpD and rP1-C antigen combination were further assessed by binary logistic regression analysis. We showed that combination of rAtpD and rP1-C discriminated maximally between the patients infected with M. pneumoniae (children and adults) and the healthy subjects for the IgM class, performing better than the single recombinant antigens or the commercial whole-cell extract. CONCLUSION: These results suggest that AtpD can be used as an antigen for the immunodiagnosis of early and acute M. pneumoniae infection in association with adhesin P1, providing an excellent starting point for the development of point-of-care diagnostic assays.
Subject(s)
Bacterial Proteins/immunology , Gene Expression , Mitochondrial Proton-Translocating ATPases/immunology , Mycoplasma pneumoniae/enzymology , Pneumonia, Mycoplasma/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Proteins/genetics , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Mitochondrial Proton-Translocating ATPases/genetics , Mycoplasma pneumoniae/genetics , Mycoplasma pneumoniae/immunology , Pneumonia, Mycoplasma/diagnosis , Pneumonia, Mycoplasma/microbiology , Protein Subunits/genetics , Protein Subunits/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Young AdultABSTRACT
Because of the wide distribution of the survivin Ag in a variety of tumors, we have investigated the survivin-specific CD4+ T cell response in healthy donors and cancer patients. Screening of the entire sequence of survivin for HLA class II binding led to the identification of seven HLA-DR promiscuous peptides, including four HLA-DP4 peptides. All of the peptides were able to prime in vitro CD4+ T cells of eight different healthy donors. The peptide-specific T cell lines were stimulated by dendritic cells loaded with the recombinant protein or with the lysates of tumor cells. The high frequency of responders (i.e., immunoprevalence) was provided by a wide reactivity of multiple peptides. Six peptides were T cell stimulating in at least half of the donors and were close to CD8+ T cell epitopes. HLA-DR molecules were more frequently involved in T cell stimulation than were HLA-DP4 molecules, and hence immunoprevalence relies mainly on HLA-DR promiscuity in the survivin Ag. In two cancer patients a spontaneous CD4+ T cell response specific for one of these peptides was also observed. Based on these observations, the tumor-shared survivin does not appear to be the target of immune tolerance in healthy donors and cancer patients and is a relevant candidate for cancer vaccine.
