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.     

Evaluation of a new commercial microimmunofluorescence test for detection of antibodies toChlamydia pneumoniae, Chlamydia trachomatis, andChlamydia psittaci

A new commercial test for chlamydial serology, the MRL-Micro-Immunofluorescent Test (MRL; MRL Diagnostics, USA) was compared with the standard microimmunofluorescence test (MIF) using sera from 246 patients.Chlamydia pneumoniae immunoglobulin G (IgG) antibodies were detected in 46.3% (MIF) and 64.2% (MRL) of sera andChlamydia trachomatis IgG in 23.2% (MIF) and 25.2% (MRL);Chlamydia psittaci IgG antibodies were found with the MRL in 1 % of the sera from a general population and in 17.3% of preselected sera with elevated complement fixation titers. Titers were usually higher with the MRL. IgG titers of 1512 were detected in only 2% of sera using the standard MIF but in 30% using the MRL. In 16 sera from threeChlamydia pneumoniae culturepositive patients, the diagnosis of acute infection could be confirmed serologically in one with the MRL test but in none with the MIF test, indicating a higher sensitivity of the MRL.     

Fallopian tube obstruction as a sequela toChlamydia trachomatis infection

The association of tubal infertility and ectopic pregnancy with Chlamydia trachomatis infection was investigated using a case-control study design. Although culture methods failed to document active chlamydial infection in the majority of cases, serology revealed a significant association of Chlamydia trachomatis antibody with tubal infertility and ectopic pregnancy. Thirteen of 18 (72 %) women with tubal factor infertility and 18 of 32 (56 %) women with ectopic pregnancy had antibodies to Chlamydia trachomatis as compared to 11 of 49 (22 %) normal pregnant controls. Interestingly, only 7 of 18 (39 %) infertile women and 5 of 36 (14 %) women with ectopic pregnancy recalled a history of pelvic inflammatory disease. These results suggest that chlamydia-associated salpingitis, whether clinically evident or subclinical, is a major contributor to diseases of tubal dysfunction.     

Immune response to genital chlamydial infection and influence ofChlamydia pneumoniae (TWAR) antibodies

Chlamydial IgG antibodies at a titre of at least 32 were found to occur at approximately the same frequency (76–88%) in three groups of patients who had been treated for genital chlamydial infection. Twenty-four patients who had recovered from acute salpingitis, however, had a higher geometric mean titre (GMT; 176) than 59 pregnant women (GMT 44) or 61 patients with uncomplicated genital chlamydial infection (GMT 57). The chlamydial antibody titres thus seemed to reflect the severity of inflammatory involvement. Antibodies to the new speciesChlamydia pneumoniae (previously known asChlamydia TWAR), did not influence the frequency or titre of antibodies toChlamydia trachomatis, arguing against the possibility of cross-reactivity or shared antigens. There was assumed to be no cross-immunity, since patients withChlamydia trachomatis infection did not have a lower frequency of antibodies toChlamydia pneumoniae.     

Detection of Seroconversion and Persistence of Chlamydia trachomatis Antibodies in Five Different Serological Tests

 Microimmunofluorescence (MIF), a Chlamydia trachomatis species-specific enzyme immunoassay incorporating lipopolysaccharide-extracted Chlamydia trachomatis L2 elementary bodies, two different synthetic peptide-based species-specific tests, and a recombinant lipopolysaccharide genus-specific test were performed on multiple follow-up sera (n=104 total) from 16 women with Chlamydia trachomatis-positive cervical swabs. These women included five with IgG seroconversions, five with Chlamydia trachomatis reinfections after initial therapy, and six with serologic follow-up of more than 6 years after antibiotic therapy. Of all the tests employed in this study, MIF IgG reverted earliest to negative titers, while MIF IgA was the least sensitive. The lipopolysaccharide-extracted elementary body enzyme immunoassay exhibited the closest correlation with the MIF test. The highest test sensitivity was observed in one of the synthetic peptide-based tests, which detected earliest seroconversions and longest IgG persistence. The other synthetic peptide-based test gave false-negative results in 2 of 16 women and did not detect seroconversion earlier than the MIF test. Seroconversion and persistence of genus-specific IgG – cross-reactivity with Chlamydia pneumoniae– against lipopolysaccharide were similar to species-specific IgG. A significant serologic response to reinfection was observed only in women with signs of pelvic inflammatory disease. Species-specific tests of high sensitivity and reproducibility are best suited for gynecological diagnostic purposes.     

Respiratory infection withchalmydia pneumoniae in middle-aged and older adult outpatients

This study was undertaken to characterize the epidemiology and clinical presentation of infection withChlamydia pneumoniae in a population composed primarily of middle-aged and older adults. Pharyngeal swabs and acute and convalescent phase sera were obtained from outpatients presenting with signs and symptoms of an acute respiratory infection. Sera were examined using the micro-immunofluorescence (MIF) test to detect antibody toChlamydia pneumoniae and complement fixation tests to detectMycoplasma pneumoniae, influenza A virus, influenza B virus, respiratory syncytial virus and adenovirus. Pharyngeal swab specimens were cultured forChlamydia pneumoniae and tested forChlamydia pneumoniae by the polymerase chain reaction (PCR). A total of 743 patients with a mean age of 40.5 ± 16.1 years were enrolled in the study. Twenty-one patients were serologically positive for acuteChlamydia pneumoniae infection in the MIF test. PCR was positive in 15 of the 20 serologically positive patients tested. AcuteChlamydia pneumoniae infection was identified in 3 % (2/76) of subjects with pneumonia, 5 % (12/247) of those with bronchitis, 5 % (3/61) of those with sinusitis only and 2 % (2/103) of those with pharyngitis only. Of the 21 patients withChlamydia pneumoniae infection, seven (mean age of 33 years) had an antibody pattern suggesting a primary infection while 14 (mean age of 54 years) had a reinfection pattern. Patients with reinfection had milder disease than those with primary infection. PCR testing in the current study confirms the previously proposed serologic criteria of acuteChlamydia pneumoniae infection.     

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).     

Acute lower respiratory tract infection associated withChlamydia pneumoniae in Germany

Serum specimens from 223 patients with acute lower respiratory tract infection were examined for antibodies toChlamydia pneumoniae using the microimmunofluorescence test. Antibodies toChlamydia pneumoniae were detected in 18 (20 %) of 91 children and 64 (48 %) of 132 adults. Among those individuals, 4 (4 %) children and 15 (11 %) adults had elevated IgG antibody titres indicating acute or recent infection. Specific IgM antibodies were observed in two patients. These results suggest that a significant proportion of lower respiratory tract infections in Germany is caused byChlamydia pneumoniae.     

Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients

Chlamydia pneumoniae has been associated to coronary artery disease by various methods including recovery of viable bacteria from plaques. The pathogenetical relevance of this is unclear but investigation of antichlamydial therapy in coronary arteriosclerosis is already in progress. The microimmunofluorescence test (MIF), the only species-specific serological assay available, might be considered useful in identifying patients with vascular chlamydial infection. However, this has never been systematically examined. We compared levels of C. pneumoniae antibodies in sera using MIF with direct detection of C. pneumoniae in coronary artery segments from 158 patients undergoing myocardial revascularization. A polymerase chain reaction (PCR) protocol, recently evaluated for use with vascular materials, detected C. pneumoniae infection in 34 patients. Correlation of serology and PCR was poor: in relation to PCR, MIF-IgG analysis had 21% sensitivity, 90% specificity, 37% positive predictive value, and 81% negative predictive value for detection of chlamydial presence. Thus, the MIF test currently appears not suitable to predict individual vascular C. pneumoniae infection. Received: 19 June 1998     

Detection of Chlamydia pneumoniae DNA in Buffy-Coat Samples of Patients with Abdominal Aortic Aneurysm

 Recent studies have suggested that Chlamydia pneumoniae infection is a risk factor for abdominal aortic aneurysm. This study explores the presence of Chlamydia pneumoniae DNA in buffy-coat samples of control subjects and of patients with abdominal aortic aneurysm. The seroepidemiological association between abdominal aortic aneurysm and Chlamydia pneumoniae was also investigated. Buffy-coat samples and serum specimens were obtained from 88 patients and 88 control subjects. Detection of Chlamydia pneumoniae DNA in buffy-coat samples and measurement of IgG antibodies to Chlamydia pneumoniae in serum specimens were performed by polymerase chain reaction and microimmunofluorescence, respectively. Chlamydia pneumoniae DNA was detected in buffy-coat samples of 18 (20%) patients and 8 (9%) control subjects (adjusted odds ratio 2.9, 95% confidence interval 1–8.5). IgG antibodies to Chlamydia pneumoniae were detected in 85 (97%) patients and 71 (81%) control subjects (adjusted odds ratio 7.2, 95% confidence interval 1.7–31). The results show an association between abdominal aortic aneurysm and either the presence of Chlamydia pneumoniae DNA in buffy-coat samples or IgG antibodies to Chlamydia pneumoniae. These findings support the hypothesis that previous infection with Chlamydia pneumoniae might be a risk factor for abdominal aortic aneurysm.     

Prevalence of antibodies toChlamydia pneumoniae in a pediatric hospital population in Belgium

To evaluate the prevalence of antibodies toChlamydia pneumoniae in a pediatric hospital population, 207 patients admitted to the pediatric unit of the hospital during the period January to April 1989 were screened. A micro-immunofluorescence test was used to measure bothChlamydia pneumoniae specific total antibody and IgM antibody. Fifty-eight (28 %) patients were found to have antibodies toChlamydia pneumoniae. Only one serum contained specific IgM. Analysis of the age specific prevalence of antibody showed a sudden rise in the 10 to 12 year old age group. Cross-reaction in the test with the otherChlamydia species is discussed. It is concluded that the incidence of primaryChlamydia pneumoniae infection during the study period was low, but that the infection occurs in Belgium with about the same prevalence as in other countries.     

Performance of Three Microimmunofluorescence Assays for Detection of Chlamydia pneumoniae Immunoglobulin M, G, and A Antibodies

The microimmunofluorescence (MIF) test is considered the “gold standard” for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different C. pneumoniae strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute C. pneumoniae infection rests on the presence of IgM antibodies, not on their level.     

Comparison of five commercial serological tests for the detection of anti-Chlamydia trachomatis antibodies

Screening for Chlamydia trachomatis-specific antibodies is valuable in investigating recurrent miscarriage, tubal infertility and extrauterine pregnancy. We compared here the performance of immunofluorescence (IF) to four other commercial tests in detecting IgG antibodies directed against C. trachomatis: two enzyme-linked immunosorbent assays (ELISAs) using the major outer membrane protein (MOMP) as the antigen, commercialised respectively by Medac and R-Biopharm (RB), one ELISA using the chlamydial heat shock protein 60 (cHSP60) as the antigen (Medac), as well as a new automated epifluorescence immunoassay (InoDiag). A total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all five tests. The prevalence of C. trachomatis-specific IgG antibodies as determined by the IF, cHSP60-Medac, MOMP-Medac, MOMP-RB and InoDiag was 14.3, 23.2, 14.3, 11.9 and 26.2%, respectively. InoDiag exhibited the highest sensitivity, whereas MOMP-RB showed the best specificity. Cross-reactivity was observed with C. pneumoniae using IF, MOMP-RB and InoDiag, and Parachlamydia acanthamoebae using the cHSP60 ELISA test. No cross-reactivity was observed between C. trachomatis and the other Chlamydiales (Neochlamydia hartmannellae, Waddlia chondrophila and Simkania negevensis). Given its high sensitivity, the new automated epifluorescence immunoassay from InoDiag represents an interesting alternative. The MOMP-based ELISA of R-Biopharm should be preferred for large serological studies, given the high throughput of ELISA and its excellent specificity.     

A simple ELISA capable of distinguishing between IgG antibodies to Chlamydia trachomatis and Chlamydia pneumoniae

Simple assays which reliably distinguish between past infection with Chlamydia pneumoniae and with Chlamydia trachomatis are needed. We developed an enzyme-linked immunosorbent assay (ELISA) for this purpose by reducing antigen cross reactive lipopolysaccharide epitopes with deoxycholate treatment and releasing antibodies of low affinity with a 6 M urea wash step. Paired serum samples from 212 patients with non-C. pneumoniae-associated community acquired pneumonia and single serum samples from 61 healthy students, 61 women with gynaecological complaints, and 100 blood donors were tested in the assay system. Excellent positive/negative correlation with the microimmunofluorescence (MIF) test was shown. This urea ELISA assay may be useful in assessment of past infection with these organisms, especially in epidemiological studies.     

Comparison of Two Commercial Microimmunofluorescence Kits and an Enzyme Immunoassay Kit for Detection of Serum Immunoglobulin G Antibodies to Chlamydia pneumoniae

We compared the MRL and the Labsystems Chlamydia pneumoniae microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to C. pneumoniae. The correlation between the results obtained with these two MIF kits was excellent (r = 0.95; P = 0.001). The cross-reactivity of the C. pneumoniae-positive sera with C. trachomatis- and C. psittaci-positive sera was assessed for each MIF kit. For C. pneumoniae-positive sera with titers of ≥32, the Labsystems MIF kit exhibited more cross-reactivity to C. psittaci than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to C. pneumoniae (for the MRL MIF kit, r = 0.79 [P = 0.001]; for the Labsystems MIF kit, r = 0.78 [P = 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of C. pneumoniae antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation.     

Immunology: Chlamydial immunoglobulin IgG and IgA antibodies in serum and semen are not associated with the presence of Chlamydia trachomatis DNA or rRNA in semen from male partners of infertile couples

The role of Chlamydia (C.) trachomatis in male infertility iscontroversial. The objective of this study was to determinethe prevalence of asymptomatic C.trachomatis infections in malepartners of infertile couples, and to compare this result withthe presence of chlamydial antibodies in serum and semen. C.trachomatiswas detected in five of 50 semen specimens (10%) by either polymerasechain reaction for C.trachomatis DNA or direct DNA probing forC.trachomatis rRNA. There was no association between the detectionof C.trachomatis in semen and the presence of chlamydial antibodiesin serum or semen. Chlamydial serum antibodies were neitherassociated with antisperm serum antibodies nor with pathologicalstandard semen parameters. These results indicate that the assessmentof chlamydial immunoglobulin IgG and IgA antibodies in serumor semen is of limited use in male infertility work-up, in contrastto its significance in female tubal infertility. The presenceof C.trachomatis in semen emphasizes the potential risk of transmissionduring artificial insemination and other assisted reproductivetechniques, and underlines the importance of sensitive directdetection methods in this group of patients.     

Chlamydia pneumoniae and screening for tubal factor subfertility

Chlamydia antibody testing (CAT) by micro-immunofluorescence (MIF) tests has been introduced into the fertility work-up as a screening test for tubal factor subfertility. In this study the role of C. pneumoniae antibodies, as a cause for false positive CAT results due to cross-reactivity with C. trachomatis antibodies in the MIF test, has been evaluated. In 240 subfertile women serological data were compared to laparoscopy findings. The prevalence of C. pneumoniae antibodies using enzyme-linked immunosorbent assay (ELISA) was 75% and did not differ between patients with and without tubal pathology. C. pneumoniae antibodies were found in 87% of women with a positive MIF test (> or =32), and in 66% with a negative MIF test (P < 0.0005). Using ELISA instead of MIF for the detection of C. trachomatis antibodies, C. pneumoniae antibodies were found in 87% of C. trachomatis positive women, and in 69% of C. trachomatis negative women (P < 0.0005). Patients without tubal factor subfertility but a positive MIF test showed C. pneumoniae antibodies more frequently than patients without tubal factor subfertility and a negative MIF test. Therefore it was suggested that C. pneumoniae antibodies may be the cause of false positive CAT results. Remarkably, tubal pathology was more common in patients who had antibodies to both C. trachomatis and C. pneumoniae.     

Previously undetected Chlamydia trachomatis infection, immunity to heat shock proteins and tubal occlusion in women undergoing in-vitro fertilization.

The relationship between a previously undetected Chlamydia trachomatis infection, tubal infertility, immunity to heat shock proteins and subsequent in-vitro fertilization (IVF) outcome was evaluated. Women with tubal occlusion, with or without hydrosalpinges, and no history of C. trachomatis infection were tested for circulating antibodies to the human 60-kDa heat shock protein (Hhsp60), the C. trachomatis 10-kDa heat shock protein (Chsp10) and C. trachomatis surface antigens prior to their initial IVF cycle. Sera were obtained from 50 women whose male partners were infertile, 58 women with tubal occlusion but no hydrosalpinx and 39 women with tubal occlusions plus hydrosalpinx. Clinical pregnancies were documented in 68% of the women with male factor infertility. This was higher than the 43.1% rate in women with tubal occlusions (P = 0.04) and the 41% rate in women with hydrosalpinx (P = 0.02). C. trachomatis antibodies were present in one (2%) women with male factor infertility as opposed to 15 (25.9%) women with tubal occlusion (P = 0.003) and 13 (33%) with hydrosalpinx (P < 0.0001). Antibodies to Chsp10 were more prevalent in women with hydrosalpinx (46.8%) than in women with male factor infertility (P < 0.0001, 6%) or tubal occlusion (P = 0.0009, 15.5%). Hhsp60 antibodies were equally more prevalent in women with tubal occlusion plus (46.8%) or minus hydrosalpinx (41.4%) than in women with male factor infertility (P < 0.0002). Hhsp60 was more prevalent in those women positive for Chsp10 (P = 0.02) or C. trachomatis (P = 0.04) antibodies than in women lacking these antibodies. There was no relationship between any of the antibodies measured in sera and IVF outcome.     

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.     

Relationship betweenChlamydia trachomatis infection and elevated serum immunoglobulin M levels in premature infants

Serum immunoglobulin M (IgM) antibodies to Chlamydia trachomatis and to human cytomegalovirus (CMV) were detected by enzyme-linked fluorescence assay and enzyme-linked immunosorbent assay, respectively in 19 premature infants with chronic lung diseases, in 43 extremely low birth weight premature infants and in 123 neonates with elevated serum IgM levels. Ten of the 19 premature infants with chronic lung diseases had elevated serum IgM levels, and five had IgM antibodies to Chlamydia trachomatis.Three of the 43 extremely low birth weight premature infants had elevated serum IgM levels, and two had IgM antibodies to Chlamydia trachomatis.Three of the 123 neonates with elevated serum IgM levels (excluding those with chronic lung diseases and extremely low birth weight) had IgM antibodies to CMV. These results suggest that chronic lung diseases in low birth weight infants might be caused by intrauterine Chlamydia trachomatis infection.