Evaluation of Chlamydia pneumoniae 43- and 53-kilodalton recombinant proteins for serodiagnosis by Western Blot.

Chlamydia pneumoniae is a common cause of respiratory infection. It has also been shown to be associated with coronary heart disease. Two proteins that have been reported to be recognized frequently during human infection are proteins having molecular masses of 43 and 53 kDa. In order to develop a useful alternative serological test to the microimmunofluorescence (micro-IF) assay, recombinant 43-kDa and 53-kDa chlamydia-specific proteins were evaluated in dot blot and/or for comparison to the standard micro-IF test. Primers for amplification were derived from genome sequence information for two C. pneumoniae genes (CPn0809 and CPn0980) encoding 53-kDa proteins and four C. pneumoniae genes (CPn0562, CPn0927, CPn0928, and Cpn0929) encoding 43-kDa proteins of unknown function, which were Chlamydia specific and not found in Chlamydia trachomatis. The 53-kDa protein product of CPn0809 or the N-terminal 18-kDa portion had better specificity than any of the 43-kDa recombinants but was much less sensitive than micro-IF. In contrast, the 53-kDa protein encoded by CPn0980 was recognized by 11 of 12 (92%) acute-phase sera, 35 of 46 (76%) chronic sera, 0 of 12 micro-IF-negative sera (C. pneumoniae and C. trachomatis negative), and 1 of 12 (8%) C. pneumoniae negative, C. trachomatis positive sera. Thus, it appears that the 53-kDa protein encoded by CPn0980 has potential use for serodiagnosis of C. pneumoniae infection.     

Characterization and intracellular localization of putative <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> effector proteins

We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation.     

Host cell Golgi anti-apoptotic protein (GAAP) and growth of Chlamydia pneumoniae

Chlamydia pneumoniae protein CPn0809 is a type three secretion system substrate, the exact function of which in infection pathogenesis has remained unknown. In this study, we identified by yeast two-hybrid screening a potential host cell interaction partner of CPn0809, Golgi anti-apoptotic protein (GAAP), a conserved protein found in eukaryotic cells. GAAP gene is expressed at relatively constant levels and its expression remained stable also after C. pneumoniae infection. The interaction between GAAP and C. pneumoniae was suggested by transfection studies. GAAP knock-down by siRNA in infected A549 cells resulted in an increased number of C. pneumoniae genomes and growth of the bacteria as judged by quantitative PCR and inclusion counts, respectively. Silencing of GAAP did not make the A549 cells more susceptible to apoptosis per se, and infection with C. pneumoniae prevented staurosporin-induced apoptosis also in transfected cultures. Taken together, the proposed interaction between C. pneumoniae and GAAP modulates bacterial growth in A549 cells.     

Identification of Two Novel Genes Encoding 97- to 99-Kilodalton Outer Membrane Proteins of Chlamydia pneumoniae

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those of putative outer membrane proteins encoded by the Chlamydia psittaci and Chlamydia trachomatis gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy showed that epitopes of Omp4 were exposed on the surface of C. pneumoniae elementary bodies, reticulate bodies, and outer membrane complex. Proteins encoded by the C. pneumoniae gene family seem to be dominant antigens in experimentally infected mice.     

Molecular Cloning and Characterization of the Genes Coding for the Highly Immunogenic Cluster of 90-Kilodalton Envelope Proteins from the Chlamydia psittaci Subtype That Causes Abortion in Sheep

Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype of Chlamydia psittaci that causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The four pomp genes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected in C. psittaci avian and feline pneumonitis strains, respectively. No pomp homologs were found in strains of C. trachomatis and C. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes of C. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.     

Analysis of the Humoral Immune Response to Chlamydia pneumoniae by Immunoblotting and Immunoprecipitation

Chlamydia pneumoniae is a widely spread agent of respiratory tract infections in humans. A reliable serodiagnosis of the disease is hampered by the poor knowledge about immunodominant antigens in C. pneumoniae infections. We applied a novel strategy to identify immunogenic proteins of C. pneumoniae TW183 combining metabolic radiolabeling of de novo-synthesized chlamydial antigens with immunoprecipitation. By this technique C. pneumoniae antigens of approximately 160, 97 to 99, 60 to 62, 40, 27, and 15 kDa were detected in the vast majority of sera from patients with a current C. pneumoniae infection. By immunoblotting purified elementary bodies of C. pneumoniae TW183 with the same sera, only the 60- to 62-kDa antigen could be detected consistently. Sequential immunoprecipitation performed at different stages of the chlamydial developmental cycle revealed that the 60- to 62-kDa antigen is strongly upregulated after 24 to 48 h of host cell infection and is presented as a major immunogen in both C. pneumoniae-infected patients and mice. We conclude that, due to its high sensitivity and concurrent preservation of conformational epitopes, metabolic radiolabeling of chlamydial antigens combined with immunoprecipitation may be a useful method to reveal important immunogens in respiratory C. pneumoniae infection which might have been missed by immunoblot analysis.     

Human Mannose-Binding Protein Inhibits Infection of HeLa Cells by Chlamydia trachomatis

The role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L₂/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L₂ was not affected by supplementation with complement or addition of an L₂-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L₂, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.     

High-Level Expression of Chlamydia psittaci Major Outer Membrane Protein in COS Cells and in Skeletal Muscles of Turkeys

The omp1 genes encoding the major outer membrane proteins (MOMPs) of avian Chlamydia psittaci serovar A and D strains were cloned and sequenced. The nucleotide sequences of the avian C. psittaci serovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avian C. psittaci serovar A strain 6BC, 84.6 and 99.8% identical to that of the avian C. psittaci serovar D strain NJ1, 79.1 and 81.1% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of the Chlamydia trachomatis L2 strain, and 57.5 and 60.4% identical to that of the Chlamydia pneumoniae IOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.     

Evaluation of serological methods in the diagnosis ofChlamydia pneumoniae pneumonia during an epidemic in Finland

A complement fixation (CF) test, a micro-immunofluorescence (micro-IF) test and an enzyme immunoassay (EIA) using Re-lipopolysaccharide as antigen were compared in the diagnosis of chlamydial infection in 136 mainly elderly patients hospitalized with community-acquired pneumonia during aChlamydia pneumoniae epidemic in Finland in 1986–1987. Chlamydial pneumonia was diagnosed in 58 (42.6 %) of the 136 pneumonia patients; 44 (75.9 %) of them could be shown by micro-IF to be caused byChlamydia pneumoniae, three byChlamydia psittaci and four byChlamydia spp. Only 5 (11.4 %) of 44 patients withChlamydia pneumoniae pneumonia were IgM-positive, indicating that the majority of cases were reinfections. In this population of mainly elderly patients the CF test was insensitive, being positive in only 6 (10.3 %) of 58 cases of chlamydial pneumonia. The EIA detected 72.4 % of cases and micro-IF 87.9 % of cases (including infections withChlamydia pneumoniae, Chlamydia psittaci andChlamydia spp.). In the EIA 77 % of positive cases were positive in serum samples taken a week apart, whereas the corresponding figure for micro-IF was 50 %. In micro-IF the measurement of IgA antibody levels is recommended and IgM-positive sera should be retested after removal of IgG antibody to avoid false-positive findings due to presence of rheumatoid factor. The collection of a third serum sample, for instance one month after onset, is also recommended, since half of the patients showed a diagnostic response in the micro-IF only in the sera taken one month apart.     

Inclusion Fluorescent-Antibody Test as a Screening Assay for Detection of Antibodies to Chlamydia pneumoniae

A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the “gold standard.” In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of ≥16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of ≥512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (≥16) or elevated (≥512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).     

Analysis of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2_aa23-aa93, Chlamydia psittaci-Omp2_aa23-aa94, and Chlamydia trachomatis-Omp2_{aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547}). By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P 0.0001). The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.     

Immune response toChlamydia trachomatis heat-shock protein in infertile female patients and influence ofChlamydia pneumoniae antibodies

A total of 446 sera from 245 patients with primary or secondary infertility, all of whom were examined laparoscopically, 117 patients withChlamydia trachomatis-positive cervical swabs, and 84 control persons (50 obstetric patients and 34 female blood donors) were tested for antibodies toChlamydia trachomatis and toChlamydia pneumoniae with the microimmunofluorescence (MIF) test. MIF test antibody rates were highest in patients with complete tubal occlusion (73%) and in patients with provenChlamydia trachomatis infection (74%), whereas only 9 to 10% of the control group showedChlamydia trachomatis antibodies. Reaction to the 60 kDa antigen ofChlamydia trachomatis, a heat-shock protein (hsp) analogue, has been suggested as a possible marker for the development of chronic sequelae afterChlamydia trachomatis infection. Immunoblot analysis of 222 sera (169 infertility patients, 20 antigen-positive patients, and 33 mothers) showed a significantly higher anti-hsp antibody rate in patients with complete tubal occlusion than in infertility patients with normal fallopian tubes (76% vs. 19%, p<0.001). The presence of antibodies not only toChlamydia trachomatis but also toChlamydia pneumoniae in the MIF test was associated with a significantly higher rate of anti-hsp antibodies and with complete tubal occlusion. This association did not appear to be due to cross-reactivity betweenChlamydia pneumoniae and Chlamydia trachomatis antibodies in the MIF test.     

Identification ofChlamydia pneumoniae-specific protein antigens in immunoblots

The immunoblot patterns of 248 sera, all examined previously by the microimmunofluorescence test (MIF) for species-specificChlamydia antibodies, were analyzed. Predominant specific antibody activity was directed to the 54 kDa protein ofChlamydia pneumoniae, which was recognized by 93 % of sera positive forChlamydia pneumoniae by MIF but by only 2 % of sera positive forChlamydia trachomatis and negative forChlamydia pneumoniae and by 3 % of sera negative for bothChlamydia pneumoniae andChlamydia trachomatis. This antigen appears to be specific forChlamydia pneumoniae. OtherChlamydia pneumoniae-specific protein antigens were recognized far less frequently. Absorption analysis indicated that the 54 kDa protein is located on the surface of theChlamydia pneumoniae elementary bodies.     

Pgp3 Antibody Enzyme-Linked Immunosorbent Assay,a Sensitive and Specific Assay for Seroepidemiological Analysis of Chlamydia trachomatis Infection

Understanding of the burden of Chlamydia trachomatis infection and its clinical sequelae is hampered by the absence of accurate, well-characterized tests using serological methods to determine past exposure to infection. An “in-house” immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) based on the C. trachomatis-specific antigen Pgp3 was produced and evaluated against three commercial ELISAs derived from the major outer membrane protein: the Medac pELISA plus, the Savyon SeroCT-IgG ELISA, and the Ani Labsystems IgG enzyme immunoassay. Sensitivities and specificities were determined using sera from both male and female patients (n = 356) for whom C. trachomatis had been detected in the lower genital tract at least 1 month prior to the testing of the sample and from 722 Chlamydia-negative children aged 2 to 13 years. The Pgp3 ELISA was significantly more sensitive (57.9% [95% confidence interval {95% CI}, 52.7 to 62.9%]) than the Ani Labsystems (49.2% [95% CI, 44.0 to 54.3%]; P = 0.003), SeroCT (47.2% [95% CI, 42.1 to 52.4%]; P < 0.0005), and Medac (44.4% [95% CI, 39.3 to 49.6%]; P < 0.0005) ELISAs. The Pgp3, Ani Labsystems, and SeroCT assays, but not the Medac assay, had significantly higher sensitivity for female specimens than for male specimens (73.8 versus 44.2%, 59.8 versus 40.5%, 55.5 versus 40%, and 45.7 versus 43.7%, respectively). For female patients, the Pgp3 assay was 14.0% (95% CI, 5.5 to 22.5%) more sensitive than the next most sensitive ELISA, the Ani Labsystems assay (P = 0.001). There was no significant difference in specificity between the Pgp3 (97.6% [95% CI, 96.2 to 98.6%]), Ani Labsystems (99% [95% CI, 97.7 to 99.6%]), SeroCT (97.2% [95% CI, 95.7 to 98.2%]), and Medac (96% [95% CI, 94.3 to 97.2%]) ELISAs. None of the ELISAs showed evidence of cross-reactivity with antibodies to Chlamydia pneumoniae.Chlamydia trachomatis is the commonest sexually transmitted bacterial infection in developed countries, with national surveillance programs consistently showing rising rates of diagnosed infections over the past decade. In the United Kingdom, figures based on cases diagnosed in departments of genitourinary medicine (GUM) suggest a population rate of 190 per 100,000 men and 187 per 100,000 women (52). Reported rates are highly dependent on the level of testing at different clinics, with the probability that many Chlamydia cases are not diagnosed. The population prevalence of uncomplicated genital Chlamydia in 16- to 24-year-olds in the United Kingdom is thought to be between 2% and 6% in both men and women (17, 33), while the opportunistic National Chlamydia Screening Programme (2008) indicates a higher prevalence of around 10%, likely due to selective testing of higher-risk individuals (15). Nucleic acid amplification tests, commonly used in GUM clinics, identify infection only when the organism is present. Once infection has been resolved, these tests provide no information on past exposure. While detection rates are rising, due in part to increased screening and testing, the overall prevalence of past C. trachomatis exposure is not known.The prevalence of past exposure to genital C. trachomatis and changes over time in age-specific prevalence can be explored serologically. For instance, in Finland, Lyytikäinen et al. (32) studied pregnant women under the age of 29 using a commercial enzyme-linked immunosorbent assay (ELISA) based on C. trachomatis-specific peptides derived from the major outer membrane protein (MOMP). However, for wider application, confidence in the sensitivity and specificity of available antibody tests is critical (24). None of the current ELISAs have ever been rigorously evaluated against large numbers of well-defined serologically positive and negative control sera; hence, their sensitivity and specificity remain open to question.Chlamydia trachomatis is from the same family, Chlamydiaceae, as Chlamydia pneumoniae, a common respiratory pathogen with which it shares genetic homology (26). Sera from patients exposed to C. trachomatis show diverse serological profiles against immunodominant C. trachomatis antigens (2, 18, 42, 47, 49), many of which are cross-reactive with sera from patients exposed to other chlamydial species, in particular C. pneumoniae (2, 5, 19, 44). In addition, C. trachomatis antigens, such as the 60-kDa heat shock protein (hsp60) and lipopolysaccharide (LPS), cross-react with other bacterial species (35, 50, 57). Microimmunofluorescence (MIF) (54), which detects antibodies to chlamydial elementary bodies (EB), has long been considered the “gold standard” for the serodiagnosis of chlamydial infections (55). However, the procedure lacks standardization and is subjective; moreover, its specificity is considered suspect because of cross-reactivity with other chlamydial species (5, 27, 40, 44). A number of ELISAs are also commercially available, including several based on peptides of MOMP, which makes up 60% of the total outer membrane protein and is highly immunogenic (8).In addition to MOMP, the Pgp3 protein, expressed by open reading frame 5 of the chlamydial plasmid and secreted into the host cell cytosol, is a promising C. trachomatis-specific immunogen (11, 31), since the plasmid is rarely found in C. pneumoniae isolates (51), and its sequence is highly conserved (<1% divergence) between strains (7, 10, 22). Sensitivities of 50 to 60% and specificities of 80 to 90% have been reported for Pgp3 ELISAs when sera from acutely C. trachomatis infected patients and from healthy blood donors, respectively, have been assayed (1-3).The aim of this study was to produce a sensitive and specific C. trachomatis Pgp3 ELISA for fast throughput of large numbers of sera, to be used particularly in epidemiological studies and potentially as a method for assessing the population impact of Chlamydia screening programs (24). Its performance was evaluated against those of three commercially available ELISAs using well-characterized sera from people who have or have never been exposed to C. trachomatis.     

Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital Chlamydia trachomatis Infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

Chlamydia trachomatis IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates

Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.     

Characterization of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2 in Chlamydial Infection

Detection of antibodies to an outer membrane protein 2 (OMP2) by enzyme-linked immunosorbent assay (ELISA) by using either the Chlamydia trachomatis- or the Chlamydia pneumoniae-specific protein was investigated. OMP2 is an immunodominant antigen giving rise to antibody responses in humans infected with different C. trachomatis serovars (A to C and D to K) or with C. pneumoniae, which could be detected by OMP2 ELISA. OMP2 ELISA is not species specific, but antibody titers were usually higher on the homologous protein. The sensitivity of this assay was high but varied according to the “gold standard” applied. Levels of antibody to C. pneumoniae OMP2 as detected by ELISA seem to return to background or near-background values within a shorter period of time compared to antibodies to C. pneumoniae detected by microimmunofluorescence (MIF), making it more likely that positive results in ELISA reflect recent infection. Thus, OMP2 ELISA has distinct advantages over MIF and commercially available ELISAs and might be a useful tool for the serodiagnosis of chlamydial infection.     

Antibody to <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis> proteins,TroA and HtrA,as a biomarker for <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis> infection

We studied whether antibody to two chlamydial proteins (TroA and HtrA) could be used as biomarkers of Chlamydia trachomatis infection. Methods: Recombinant proteins C. trachomatis TroA and HtrA were used as antigens in enzyme immunoassay (EIA). Both IgG and IgA antibody responses were studied. Results: IgG or IgA antibody to either protein was infrequently detected in sera from healthy blood donors or virgin girls. Patients attending the STI Clinic and patients with perihepatitis had often IgG antibody against TroA (25 and 50 % respectively) and HtrA (21 and 38 % respectively). Especially in sera from patients with chlamydial perihepatitis, the A_450nm values with TroA were high (mean 1.591). A positive correlation between C. trachomatis MIF antibody and TroA (r? =?0.7) as well as HtrA (r? = 0.5) antibody was observed in sera from STI clinic patients and perihepatitis patients. Individuals with C. trachomatis infection and positive serology already when seeking medical attention had higher A_450nm values for TroA (0.638) and HtrA (0.836) than patients with no marker of previous exposure or with no infection (0.208 and 0.234 respectively). Diagnosis of genital C. trachomatis infection is often NAAT-based, whereas serology has little value in testing for uncomplicated genital C. trachomatis infection. TroA and HtrA antibodies are potential biomarkers for evaluation of ascending and repeated C. trachomatis infection.     

Inhibition of PCR in Genital and Urine Specimens Submitted for Chlamydia trachomatis Testing

We determined the frequency of PCR inhibition in genital and urine specimens submitted for Chlamydia trachomatis testing using the internal control DNA provided with the COBAS AMPLICOR C. trachomatis test and assessed methods to remove it. Inhibition occurred in 65 of 906 (7%) cervical swabs, 23 of 51 (45%) urethral swabs, and 2 of 175 (1.1%) urine samples. Overall, inhibition was eliminated in processed specimens after storage at 4°C in 77 of 90 specimens (86%), freezing at −70°C in 59 of 82 specimens (72%), storage at 4°C followed by either 1:100 dilution in 37 of 43 specimens (86%) or 1:10 dilution in 42 of 47 specimens (89%), and phenol-chloroform extraction in 79 of 80 specimens (99%). No positive specimens were missed due to inhibition. We conclude that PCR inhibition is rare with urine specimens and infrequent with endocervical swabs but occurs frequently with urethral swabs. The frequency of PCR inhibition may be significantly reduced by methods which can be easily incorporated into the processing of specimens.     

Evaluation of the Biostar Chlamydia OIA Assay with Specimens from Women Attending a Sexually Transmitted Disease Clinic

Chlamydia trachomatis infections are the most prevalent sexually transmitted diseases (STDs) in the United States. In acute-care settings such as clinics and emergency rooms, a desirable chlamydia screening assay should exhibit good sensitivity and good specificity and should provide test results while the patient is still present. The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) that provides test results in less than 30 min and that uses a test format that allows office-based testing. This assay is performed entirely at room temperature without the need for rotators or other specialized equipment. The goal of this study was to compare the performance of the Biostar Chlamydia OIA for the detection of C. trachomatis with the performance of cell culture, direct fluorescent-antibody (DFA) assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), and PCR (Roche Amplicor Chlamydia trachomatis; Roche, Branchburg, N.J.) for the detection of C. trachomatis infections in women attending an urban STD clinic. For calculations of relative test performance (sensitivity, specificity, and positive and negative predictive values), patient specimens that yielded positive results by two or more of the four assays (cell culture, DFA assay, PCR, and OIA) were classified as “true infections.” By these criteria, 42 of 306 total specimens were classified as positive for C. trachomatis (positive prevalence, 13.7%), 11 (3.6%; 10 by PCR and 1 by DFA assay) were positive by a single assay, and 253 (82.7%) were negative by all four tests. All culture-positive specimens were also positive by at least one other assay. Among the culture-negative specimens, 14 (5%) specimens were positive by two of the three non-culture-based assays used. By using the criterion that positivity by at least two of the tests indicated a true infection, the relative sensitivities were as follows: culture and PCR, 92.9% each; Biostar Chlamydia OIA, 73.8%; and DFA assay, 59.5%.Chlamydia trachomatis infections are the most prevalent sexually transmitted diseases (STDs) in the United States and are a major preventable cause of infertility, ectopic pregnancy, and chronic pelvic pain in women (3). In addition, because the signs and symptoms of infection are often mild or even absent, laboratory testing plays a central role in efforts to control chlamydia.Optimally, tests for the detection of chlamydia should be sensitive and specific and should provide results quickly to guide patient management. Until recently, the standard for the diagnosis of C. trachomatis infection has been cell culture (8, 9). However, in most clinical settings, antigen or nonamplified nucleic acid detection tests have been preferentially used for testing for chlamydia because of their logistical advantages and lower costs, despite the observation that these tests are generally less sensitive than cell culture (8). More recently, evaluations of newer nucleic acid amplification assays for C. trachomatis diagnosis such as PCR or ligase chain reaction have shown the true sensitivity of cell culture for chlamydia detection to be 65 to 85% (15, 18). Despite their greater sensitivities, amplified nucleic acid detection tests are not used in many clinical settings, in part because of their substantially higher costs compared to those of nonamplification assays. In addition, like most currently available tests for the diagnosis of C. trachomatis infection, the nucleic acid amplification tests usually do not provide test results at the time of screening but typically require turnaround times of a day or more, a characteristic that may introduce delays in translating test results into treatment (6, 16). In some settings there are demonstrable advantages to tests that can provide results while patients are still present in clinical settings, particularly if the performance of such tests is comparable to the performance of alternate tests (6, 16).The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) designed to rapidly detect chlamydia infection in women, providing test results in less than 30 min in a test format that allows office-based testing. To evaluate the performance of the Biostar Chlamydia OIA for the detection of C. trachomatis, we compared the performances of four assays, i.e., cell culture, an immunofluorescence antigen detection assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), PCR (Roche Amplicor Chlamydia trachomatis; Roche, Branchburg, N.J.), and the Biostar Chlamydia OIA, for the detection of C. trachomatis infections in women attending an urban STD clinic.