Comprehensive Molecular Serology of Human<i>Chlamydia trachomatis</i>Infections by Peptide Enzyme-Linked Immunosorbent Assays

Abstract

Sensitive species-specific detection of anti-<i>Chlamydia trachomatis</i> antibodies is compromised by cross-reactivity of the <i>C. trachomatis</i> antigens used in standard microimmunofluorescence (MIF) testing and enzyme-linked immunosorbent assays (ELISAs). Previously, we discovered 48 strongly reactive <i>C. trachomatis</i>-specific B cell epitope peptides from 21 immunodominant proteins. Here we comprehensively evaluated the 11 top-ranked <i>C. trachomatis</i>-specific peptide antigens from 8 proteins for use in <i>C. trachomatis</i> serology. Sera from 125 women with nucleic acid amplification test (NAAT)-confirmed active <i>C. trachomatis</i> infection and from 49 healthy women with a low risk of <i>C. trachomatis</i> infection were used as anti-<i>C. trachomatis</i> antibody-positive and -negative sera. Results obtained for detection of IgG1, IgG3, and IgA1 antibodies against the 11 <i>C. trachomatis</i> peptide antigens were compared to results from 4 commercial anti-<i>C. trachomatis</i> IgG ELISAs. Using composite reference standards (CRS) of all assays for anti-<i>C. trachomatis</i> antibody status, commercial ELISAs detected antibodies in antibody-positive women with sensitivities of 51.5% to 64.8%. In contrast, a combination of the results of all 11 peptides detected IgG (IgG1 and IgG3) antibodies with 91.8% sensitivity, and a labor-saving combination of the 5 optimal peptides still detected antibodies in antibody-positive women with 86.5% sensitivity (all at 98% specificity). The superior performance of the combined peptide ELISAs was confirmed by area under the receiver operating characteristic curve (ROC-AUC), likelihood ratio, and predictive value analyses. The higher sensitivity of the peptide assays results from using multiple B cell epitopes of several <i>C. trachomatis</i> immunodominant proteins, including OmpA, compared to exclusively using the OmpA antigens used in commercial ELISAs. Thus, ELISAs with combined use of synthetic peptide antigens for <i>C. trachomatis</i> antibody detection have the advantage of simultaneous high sensitivity and high specificity.<b>IMPORTANCE</b> For detection of anti-<i>Chlamydia trachomatis</i> antibodies by serological assays, use of classical whole-organism chlamydial antigens results in high cross-reactivity. These antigens bind mainly antibodies against the major outer membrane protein (OmpA) and bind antibodies against other immunodominant non-OmpA proteins to a lesser extent, resulting in poor assay sensitivity. The specificity of <i>C. trachomatis</i> serology is also compromised by the high prevalence of cross-reactive anti-<i>C. pneumoniae</i> antibodies in human populations. We previously identified 48 highly specific <i>C. trachomatis</i> B cell epitope peptide antigens of 21 immunodominant proteins. This study validated peptide antigen-based novel ELISAs that provide highly specific and sensitive detection of anti-<i>C. trachomatis</i> antibodies. Compared to four commercial ELISAs that achieved only poor sensitivities (51.5% to 64.8%), the combined signals of 5 to 11 peptides provided high sensitivity (86.5% to 91.8%) at the same 98% specificity. Thus, by using multiple peptide antigens of immunodominant proteins, we created simple ELISAs with specificity and sensitivity superior to standard <i>C. trachomatis</i> serodiagnosis.