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Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 326-331 Original Article Diagnosis and follow-up of genital chlamydial infection by direct methods and by detection of serum IgG, IgA and secretory IgA AS Fresse1, JM Sueur1, F Hamdad2 1 Departmental Laboratory of Picardie, Amiens University Hospital, France Date of Submission: 21-Dec-2009 Code Number: mb10099 PMID: 20966563 Abstract Purpose: To determine the prevalence of Chlamydia trachomatis infection in a high-risk population by direct and indirect methods and to evaluate the diagnosis of secretory immunoglobulin A (sIgA). Patients and Methods: Urethral or endocervical specimens from 78 patients (48 females and 30 males) were examined by cell culture, direct fluorescence assay, PCR Cobas Amplicor (Roche Molecular Diagnostics), and sIgA was detected by the recombinant lipopolysaccharide (LPS)-enzyme-linked immunoassay (rELISA). Serum from each patient was also obtained and analysed for the presence of IgG and IgA antibody by in-house microimmunofluorescence (MIF) and by the rELISA method (Medac, Hamburg, Germany). Results: The overall C. trachomatis prevalence determined by direct methods was 28%. The detection of sIgA antibodies was significantly higher in the group of patients with a positive direct detection (50%) than in the group of negative direct detection (10.7%). The Chlamydia-specific IgA antibodies were detected by the rELISA in 40.9 and 53.6% of group I (positive direct detection) and group II patients (negative direct detection), respectively. The species-specific IgA antibodies were detected by the MIF method in 18.2 and 16.1% of group I and II patients, respectively. Chlamydia genus-specific IgG antibodies were detected by the rELISA in 86.4 and 83.9% of group I and group II patients and, C. trachomatis specific IgG were present in 81.8 and 73.2% of group I and group II patients, respectively, as assessed by the MIF test. Conclusion: Combining the positive direct methods and/or positive sIgA antibody results from cervical or urethral specimens had an indication of current C. trachomatis infection. Keywords: C. trachomatis infections, sexually transmitted disease, direct diagnosis methods, secretory IgA, IgA and IgG antibodies in serum Introduction Chlamydia trachomatis is one of the most common sexually transmitted pathogens of humans, with an estimated 92 million new cases occurring worldwide each year. [1] Seventy to 90% of women and 50 to 70% of men with chlamydial infection are asymptomatic, and infected men and women without symptoms can serve as important reservoirs for new infections. Therefore, infected individuals may not be identifiable, and chlamydial infection in men and women may persist for longer periods. [2] Manifestations involve urethritis, cervicitis, pelvic inflammatory disease (PID), and result in ectopic pregnancy, infertility, and chronic pelvic pain in women. [3] A prolonged exposure to chlamydial antigens may result from chronic or persistent infection, and several sero-epidemiological studies have shown in women that chronic or repeated infection are a significant factor in the development of occlusive infertility or ectopic pregnancy. [4],[5] In pregnant women, chlamydial infection is associated with adverse pregnancy outcomes, including preterm delivery and postpartum endometritis; perinatal transmission to infants can cause neonatal conjunctivitis and pneumonia. [6],[7] In men, urethritis and sometimes acute epididymitis occur, and result in prostatitis and infertility. [8] An early diagnosis and appropriate treatment as well as a follow-up of the therapy are important to prevent inflammatory complications, and to limit the spread of these diseases in the community. Because Chlamydiae are obligate intracellular pathogens, the specimen collection should usually be to include the host cells that harbor the organism. The cell culture, direct detection of the bacterial antigens, and the direct immunofluorescence assay (DFA), which were until the mid 1990's the tools of direct diagnosis, have been largely replaced by the nucleic acid amplification tests (NAATs) for diagnosing or screening chlamydial infections because asymptomatically infected individuals may shed fewer organisms. [9] The NAATs are now widely accepted and recognized as the "gold standard" for the diagnosis of chlamydial infections. The immune response to many infectious diseases involves the production of secretory IgA (sIgA), which are the predominant immunoglobulin present in mucous secretions. The presence of local antibodies in genital secretions correlate well with the presence of an active infection and, their detection will pinpoint the patients that are most likely to still harbor this microorganism in their reproductive tracts [10] and, can be helpful in making decisions on therapy. Both local and systemic antibodies can be detected in C. trachomatis infection. Serology is commonly used for the diagnosis of complications in ascending chlamydial infection and is also a valuable tool in sero-epidemiological studies. The presence of C. trachomatis specific antibody is significantly associated with upper genital tract infection, particularly when the antibody is a high titer. The objective of this present study was to evaluate the direct diagnosis of C. trachomatis in a high-risk population using the cell culture, DFA and PCR Cobas Amplicor, and to evaluate the diagnosis of sIgA by the recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent (rELISA) method (Medac, Hamburg, Germany). The Chlamydia-specific IgG and IgA antibodies in sera were also detected. Two different serological techniques were used: the microimmunofluorescence (MIF) test, which is still regarded as the "gold standard" and the commercial rELISA method. Patients and Methods Patients Forty eight women and 30 men symptomatic or asymptomatic attending at one French Sexually Transmitted Disease (STD) Clinic were screened for C. trachomatis infections. The study included cases of symptomatic lower genital tract infections (urethritis for males, cervicitis and urethritis for females) and upper genital tract infections (epididymitis or prostatitis for males, salpingitis, pelvic inflammatory disease or perihepatitis for females). Some patients (eight) had already received an antibiotic treatment at the moment of enrolment in the study. Written informed consent was obtained from all individuals. All patients were asked to give their address where they could be contacted confidentially for any necessary treatment. Materials A total of 78 patients were submitted to urethral or cervical swab collections. Four swabs were collected each time for direct detection methods in the following order; the first one was placed in 1 ml transport medium (2-Sucrose-Phosphate) for cell culture. The second swab was smeared and fixed with a methanol on a slide for testing by DFA. The third one was placed in 0.5ml of phosphate-buffered saline (PBS), the liquid was extracted from the swab with a Pasteur pipette, the sample was microcentrifuged, and the secretory IgA was performed on the supernatant. Because NAATs testing is much more sensitive than the other tests, we chose always to take the NAAT swab last. The samples and slides were stored frozen at -80°C and at -20°C, respectively until further use. Blood samples for the detection of specific IgG and IgA antibodies were obtained on the same day. Eighteen patients had several follow-up examinations within one to six months. Methods The DFA test (Syva MicroTrak) was performed according to the manufacturer's instructions. This test used a monoclonal antibody raised against the major outer membrane protein of all serotypes of C. trachomatis. Positive and negative controls were included in each test series. Specimens were considered as positive if five or more particles of elementary bodies of C. trachomatis were observed. For the isolation of C. trachomatis on cell culture, the specimens thawed were vortexed vigorously and, were sonicated. One hundred microliters (100 μl) were inoculated onto a monolayer of cycloheximide-treated McCoy cells (100 μg/ml) in the wells of multi-well cell culture dishes. The well-microculture plate was then centrifuged at 1500 g for 1 hour. The supernatant was aspirated, and 500 μL of minimal essential medium with 10% fetal bovine serum and cycloheximide (1.0 μg/ml) were added. The plates were incubated at 35°C for 48-72 hours. After incubation, the cells were fixed with methanol, and stained with the in-house monoclonal anti-LPS antibody (AD5), which also reacts against the major outer membrane protein (MOMP) of genus of Chlamydia and the murine fluorescein-conjugated anti-immunoglobulin (bioMιrieux, France). Specimens were considered as positive, when at least one inclusion was observed. The PCR, Cobas Amplicor (Roche Molecular Diagnostics Systems, Meylan, France) was performed on samples thawed and vortexed. Briefly, on 100 μl of transport-tube specimen were treated with 100 μl of lysis buffer and incubated at room temperature. Two hundred microliters (200 μl) of specimen diluent were added to the lysate, mixed, and incubated for 10 minutes at room temperature. These tubes were then centrifuged at 13000 g for 5 minutes and, incubated at 95°C for 10 minute, which reduces the effects of inhibitors. [11],[12] Fifty microliters of the processed specimens were immediately transferred to A-rings containing 50 μl of PCR Master Mix for amplification. An internal control (IC) was included in the master mix to monitor inhibition and was amplified with the C. trachomatis target DNA. Amplification and detection of C. trachomatis and IC DNA were automatically performed by the Cobas Amplicor System and, the interpretation of results was performed according to the manufacturer's instructions. The sIgA was assessed on the supernatant of endocervical or urethral specimens, diluted five-fold by an enzyme immunoassay (ELISA) based on the use of a recombinant exclusively Chlamydia genus-specific fragment of the chlamydial lipopolysaccharide (LPS). The Medac rELISAs were performed according to the manufacturer's instruction. Microtiter plates coated with recombinant poly-conjugate antigens were used for the detection of antibodies with peroxidase-conjugated anti-human IgA. The presence of sIgA was determined, as suggested by the manufacturer, by the formula: Cut-off value = mean absorbance of the negative controls + 0.32. An absorbance greater than or equal to the cut-off value + 10% has been defined as being positive for secretory IgA antibodies to C. trachomatis. For the Chlamydial IgG and IgA antibodies, the slides for MIF assay was performed with three chlamydial strains prepared from infected egg yolk sacks (C. trachomatis LB1 serovar L2, C. psittaci Loth and C. pneumoniae IOL-207). Slides were fixed with acetone and were stored frozen at this stage. Serial dilutions of patient's serum were placed on the antigen dots and incubated for 30 minutes at 37°C. The slides were then washed, dried and stained with a fluorescein-conjugated anti-human class-specific Immunoglobulin (Ig) diluted at 1:100 (bioMιrieux, France). Serum IgG antibodies were tested at dilutions of 1:16 - 1:512, while serum IgA antibodies were evaluated at a dilution of 1:12 - 1:96. A titer of ≥16 without cross reactions with the antigens of the other species was considered as positive for IgG and a titer of ≥12 was considered as positive for IgA. The MIF test detects both genus-specific (LPS) antibody and species-specific major outer membrane protein (MOMP), which is the immunodominant antigen. The C. trachomatis rELISA method was performed for detection of genus-specific antibodies using a recombinant Chlamydia specific fragment of the LPS, which has not been found in any other bacterial LPS. Serum IgG antibodies were tested at dilutions of 1:100 - 1:400, while IgA were evaluated at dilutions of 1:50 - 1:400. The rELISA was performed and cut-off values were calculated according to the manufacturer's instructions. IgG cut-off value = mean absorbance of the negative control + 0.34. IgA cut-off value = mean absorbance of the negative control + 0.27. An absorbance greater than or equal to the cut-off value was defined as positive. The corresponding titer according to the cut-off index was determined according to the manufacturer's instructions. A titer of ≥100 was considered as positive for IgG and a titer of ≥50 was considered as positive for IgA. Treatment Each patient with a positive diagnosis of C. trachomatis infection received a single dose (1 gram) of azithromycin orally administered. Statistical analysis Statistical significance was evaluated by the chi-square test. Intra-group comparisons of data were performed as paired series. Confidence intervals (CI) were calculated for a risk of 0.05. Results The distribution of patients by their clinical characteristics is shown in [Table - 1]. The mean age (± SD) was: 28 years (± 3) for males and 25 years (± 3) for females. For direct C. trachomatis detection, samples were considered as positive if culture was positive or if culture was negative, but samples were positive by both DFA and PCR Cobas Amplicor. C. trachomatis was detected in 22 of the 78 patients, corresponding to a prevalence of 28% (CI, 18-38%). Two groups have been defined; the first one contains patients with positive result direct methods (22 patients). Among them, 11 (six females and five males) were positive by all direct tests (cell culture, DFA and PCR Cobas Amplicor). No PCR inhibitors were detected. The number of inclusions obtained by cell culture varies from 5 to 200 IFU/ ml. Twelve of these 22 patients had follow-up examinations and multiple samples were collected [Table - 2]. The second group contains patients with negative direct detection of C. trachomatis (56 patients). The sIgA were detected in 50% (CI, 30-70%) of group I patients and in 10.7% (CI, 4-21%) of group II patients. Among the patients with both positive direct detection method and sIgA results (n= 11), and among the patients who had several follow-up examinations (n= 6), two had negative results for both parameters within a period of 4-6 weeks following the treatment. Specific sIgA was still detected after the treatment in four cases, while the direct detection methods gave negative results [Table - 2]. In group II, one of the six patients who had negative direct detection but a positive sIgA results, revealed persistent sIgA one year later, in spite of several treatment courses. Chlamydia-specific IgA antibodies were detected by the rELISA in 40.9 and 53.6% of group I and group II patients, respectively, and by MIF method in 18.2% and 16.1% of group I and II patients, respectively. The rELISA appeared to have significantly higher sensitivity than the MIF test for the IgA determination, but the specificity was lower. Chlamydia genus-specific IgG antibodies were present in 86.4% and in 83.9% of group I and group II patients, respectively, as assessed by the rELISA method. C. trachomatis specific IgG were present in 81.8 and in 73.2% of group I and group II patients, respectively, as assessed by the MIF test. In group I, for the 12 patients who had several follow-up examinations, the IgG antibodies were positive for 10 patients by both methods. One patient was positive only by the rELISA. The IgA antibodies were positive for two patients by both methods, four other patients were positive by only the rELISA and one patient by MIF only [Table - 2]. Two IgA seroconversions have been demonstrated by rELISA, both of them occurred two weeks after the first positive direct detection. Moreover, one IgA seroconversion was observed by rELISA after a one-month course of appropriate treatment. Discussion C. trachomatis causes STD that can be treated easily and its sequelae prevented by a short course of an inexpensive antibiotic but, most infections are asymptomatic and, they remain undiagnosed so, contribute substantially to spread in the community. Laboratory methods are required for a definitive diagnosis of C. trachomatis infections; these methods include several direct and indirect techniques. The use of direct diagnosis tests with relatively lower sensitivities can result in more untreated disease and increased secondary spread of infection. The cell culture which was the only method that detects live Chlamydia was the most specific test (100%) but, the sensitivity is low (70-85% in the best experienced laboratory), and it requires specialized handling and laboratory service; it is thus reserved for specialized research laboratories and has not been generally implemented as a routine diagnosis method. [13] The DFA have been used to detect C. trachomatis in every type of specimen. One important advantage of DFA is that the quality of the specimen can be objectively assessed and remains one of the most accessible and useful diagnosis techniques available. This test is sensitive, specific and rapid, but microscopic evaluation of each specimen is laborious and requires highly trained and experienced personnel. [13] The NAATs, which have become the method of choice have high sensitivity and specificity and are considered to be a more suitable method either for screening or diagnosing chlamydial infections. [12],[14] Produced by submucosal plasma cells, C. trachomatis sIgA antibodies were reported to be a better indicator of a manifest immunoresponse caused by an antigenic stimulus than circulating antibodies. [8],[15] Studies have shown that infection of the genital tract by C. trachomatis is associated with a rise in levels of cervical sIgA and in the number of IgA-producing plasma cells. The role of sIgA is to bind to pathogens in the external environment and prevent them from binding to mucosal surfaces. [16] Therefore, local anti-chlamydial antibodies correlate with the presence of an active infection. The serology is generally not useful for the diagnosis of acute C. trachomatis genital tract infection due to the fact that antibodies elicited during infection are long-lived. Therefore, positive antibody test will not distinguish a previous infection from a current infection. However, serology may be useful in chronic C. trachomatis infection of the upper genital tract. The first immune response in acute primary chlamydial infection is the development of antibodies to genus-specific LPS, which rapidly declines. Obviously, due to the kinetics of the LPS antibodies, the LPS serology can identify recent infections without being influenced by past ones. However, the LPS antigen is common to all chlamydial species. Therefore, LPS antibody cannot distinguish between current chlamydial species infections. Generally, the serological diagnosis of active chlamydial infection is mostly based on the detection of antibodies of classes IgA. The immunoglobulin was usually detected together with IgG antibodies. It has been suggested that the presence of serum IgA antibodies, which has a half-life of about 5-7 days may be a useful marker for active C. trachomatis infection or as a marker of chronic infection with a persistent antigenic stimulation of the immune system. [8] In this study, and in this population, we found that the DFA is as sensitive as the PCR Cobas Amplicor. Indeed, all the 22 positive direct detection specimens were positive by both DFA and PCR, 11 of them were positive by cell culture as well. The high sensitivity of the DFA was probably due to specimen collection order. The sIgA were more frequently represented in direct-positive than in direct-negative patients. Indeed, the sIgA was found in 50% of the patients with a positive C. trachomatis direct detection and in 10.7% of the patients with a negative direct C. trachomatis detection. This difference was highly significant (P <10 -9 ). The absence of this local antibody in 50% of patients with proven C. trachomatis infection may be due to different immunological status, or linked to a lower versus an upper genital tract infection. As previously described, there was not always a correlation with the presence of chlamydial local antibodies and the positive direct detection of C. trachomatis.[8],[17] It has been suggested that the chlamydial concentration in the genital tract was below that required to induce immune response or that the organism may have been present in latent intracellular forms that have decreased levels of MOMP and LPS. [10] In cases of negative direct detection, in which no intercurrent treatment had been administered, the presence of sIgA may indicate a recent or current chlamydial infection where direct detection had failed. Six such cases have been observed; three of them correspond to cases of chronic upper genital tract infection, as assessed by clinical data and positive C. trachomatis specific serum IgA. The presence of serum IgA antibodies was also not always correlated with the presence of sIgA. In fact, 50 and 45% of discordance was observed in group I and group II, respectively, between rELISA-detected IgA in serum and secretions. In eight cases, sIgA appeared first and disappeared after treatment, without concomitant positive serum IgA. After a course of treatment, sIgA disappeared after 4-6 weeks, but persisted in five cases. The disappearance of sIgA after a course of treatment supported the corresponding efficacy of the therapy. The persistence of sIgA after treatment might be due to a persistent infection. The antimicrobial therapy may not eradicate the bacteria in latent or chronic forms. In group II, including cases of chronic infection, the presence of species-specific IgA, in spite of a negative direct detection, could be related in all cases to clinical manifestations of upper genital tract infections, like chronic pelvic inflammation (including one case of hydrosalpinx, as proven by hysterosalpingography). There was no significant difference between the two groups of patients concerning the prevalence of either genus-specific anti-LPS (rELISA) or MIF species-specific IgG antibodies (84.6 and 75.6%, respectively) but, the prevalence of IgA antibodies was significantly higher with the rELISA than with the species-specific MIF (P <10 -5 ) as previously described. [18] The high prevalence of MIF chlamydial IgG reflects cumulative exposure to these agents in a high-risk venereal disease population. The IgG results should always be interpreted in relation to the clinical data, because of possible cross-reactions between C. trachomatis and C. pneumoniae antigens. [11],[19] Discrepancies between the results of rELISA and MIF tests are known to occur, usually explained by the long-lasting anti-MOMP antibodies detectable by MIF, which are not detectable by the rELISA and an earlier onset and lower half-life of the LPS antibodies. There is no memory response to anti-LPS antibodies, so they can be considered as an indicator of ongoing chlamydial infection. For MIF, no conclusion could be drawn with respect to the discrimination between recent and past infections. The MIF does not seem to enable a follow-up. In conclusion, combining the positive direct methods and/or positive sIgA antibody results from cervical or urethral specimens had an indication of current C. trachomatis infection. Negative results of both the direct detection test and sIgA rELISA would confirm the absence of current infection. A reliable serological test could be of assistance in the diagnosis of C. trachomatis infection when a test for direct detection of bacteria is negative or difficult to perform, as in cases of chronic upper genital tract infections. However, a positive serum test only for IgG antibodies to C. trachomatis particularly anti-MOMP indicates that exposure to C. trachomatis has occurred. In populations with high exposure rates, each IgG value should be interpreted in connection with IgA. Positive IgG and IgA serology would confirm clinical data evocative of upper genital tract C. trachomatis infections. The IgG and IgA serology results may also provide additional elements in the follow-up, either by the absence of growing antibody titers or by the detection of lowering titers. In all cases of a confirmed genital chlamydial infection either lower or upper, successful treatment may be followed by both negative of the direct detection method and the sIgA. Due to the kinetics of the LPS antibodies and shorter time life, the IgA antibodies may be also a useful tool for a follow-up of Chlamydia infection. [20] References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10099t1.jpg] [mb10099t2.jpg] |
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