Persistence of Chlamydial Antibodies

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 23, No. 5 Persistence of Chlamydial Antibodies after Pelvic Inflammatory Disease MIRJA PUOLAKKAINEN,l* ERVO VESTERINEN,2 ESKO PUROLA,2 PEKKA SAIKKU,1 AND JORMA PAAVONEN3t Department of Virology, University of Helsinki,' and Departments I and II of Obstetrics and Gynecology, University Central Hospital,2 Helsinki, and Department of Clinical Sciences, Received 8 November 1985/Accepted 29 January 1986 University of Tampere,' Tampere, Finland The persistence of chlamydial immunoglobulin G (IgG) antibodies and long-term sequelae of pelvic inflammatory disease (PID) were studied in 70 women who had been treated for PID 3 to 6 years previously. Fifty-one women had had PID associated with Chlamydia trachomatis infection (Chlamydia group), and 19 women had had PID not associated with C. trachomatis (non-chlamydia group). Chlamydial IgG antibodies, as determined by the indirect immunofluorescence test with inclusions of C. trachomatis L2 as antigens, persisted at stable levels in 43% of the women for up to 6 years; 43% of the women showed a decrease in IgG titer, and 13% showed an increase. IgA antibody levels in serum correlated with IgG antibody levels in serum and with the presence of cervical IgA antibodies. Both serum antibodies and cervical IgA antibodies were more often found in the Chlamydia group. Forty-two percent of the women were infertile. Every fifth subsequent pregnancy was ectopic. The presence of cervical IgA antibodies might protect the women from tubal damage. Chlamydia trachomatis is now recognized as the most common sexually transmitted infectious agent. Laboratory diagnosis of the chlamydial infections can be based on isolation (17), on direct demonstration of the agent (20), or on serological methods (25). In isolation positive male urethritis seroconversions are not often encountered, since uncomplicated mucosal and superficial genital chlamydial infections do not usually produce an antigenic stimulus strong enough to elicit a readily discernible antibody response. The geometric mean titer (GMT) by the indirect immunofluorescence antibody test (IFAT) for immunoglobulin G (IgG) in urethritis is low, but moderately elevated GMTs are observed among patients with cervicitis, and clearly elevated titers are seen among patients with complicated genital infections (25). Since the early disease may be asymptomatic, antibodies have already appeared in many cases by the time serological diagnosis is attempted, hampering the demonstration of seroconversions. The high background prevalence of antibody, especially in sexually transmitted disease clinic populations, also hampers the diagnostic utility of seroconversion. In addition, chlamydial antibodies seem to persist even after the treatment of the infection, making assessment of a single IgG antibody titer difficult. The diagnosis of genital chlamydial infections cannot be based on measurement of specific IgM or IgA class antibodies, since IgM antibodies are found consistently in certain clinical conditions only (19), and the importance of serum IgA antibodies is unknown. This study was undertaken to follow long-term (up to 6.3 years) persistence of chlamydial antibodies after pelvic inflammatory disease (PID) and to evaluate the diagnostic significance of the results obtained by chlamydial serology. In addition, longterm sequelae of PID were analyzed, especially in relation to past and present serological results. * Corresponding author. t Present address: Department of Obstetrics and Gynecology, School of Medicine, University of Washington, Seattle, WA MATERIALS AND METHODS Study population. In 1983, an invitation letter for free gynecologic examination was sent to 98 women who had been treated for PID with appropriate antibiotics at the I and II Departments of Obstetrics and Gynecology, Helsinki University Central Hospital, between 1977 and The clinical diagnosis of PID was based on clinical criteria including history of lower abdominal pain of less than 3-week duration; abnormal vaginal discharge; adnexal tenderness, usually with a palpable mass; erythrocyte sedimentation rate of >15 mm/h; and fever of >38 C (11). Cervical and urethral cultures for C. trachomatis and Neisseria gonorrhoeae were performed as previously described (12). Acute- and convalescent-phase sera were obtained, and serum IgG antibodies to C. trachomatis were determined as described elsewhere in detail (16). Seventy women (71%) responded to the letter and were examined at the outpatient clinic of the same department in 1983 and Informed consent was obtained from all patients. The mean age of the women was 31.5 years (range, 20 to 48 years), and the mean follow-up time after the PID episode was 5.0 years (range, 3.1 to 6.3 years). Hospital records from the previous PID episodes were carefully reviewed, with special emphasis on microbiological findings. Clinical examination. On the follow-up visit, a detailed gynecologic and obstetric history was obtained from all women. Special attention was paid to contraceptive practices, fertility problems, and other genital symptoms. Every patient underwent a complete gynecologic examination by one of us (E.V.). Cytologic cervicovaginal (Pap) smears obtained from the posterior vaginal fornix, ectocervix, and endocervix were stained by Papanicolaou's original method. Specimens from the cervix and urethra were obtained for isolation of C. trachomatis and N. gonorrhoeae. Microbiological methods. Cycloheximide-treated McCoy cells were used for the isolation of C. trachomatis, and

2 VOL. 23, * follow up < >128 IFAT IgG titer FIG. 1. Presence of chlamydial IgG antibodies measured by LGV-IFAT. in 70 PID patients during the acute phase and on follow-up examination. growth was detected by iodine staining of the inclusions (15). Isolation of N. gonorrhoeae was performed by routine methods (10). An IFAT was carried out with inclusions of C. trachomatis L2 as antigens (lymphogranuloma venereum [LGV]-IFAT; 13). Acute-phase sera, stored at -20 C, were tested simultaneously with follow-up sera in twofold dilutions, and fluorescein-conjugated anti-human IgG (Wellcome Research Laboratories, Beckenham, England) and IgA (Kallestad Laboratories, Inc., Chaska, Minn.) were used to detect the antigen-bound antibodies. In LGV-IFAT, IgG titers.1:128 were considered high (4). Serum samples were also tested for the presence of IgG antibodies by IFAT with McCoy cell-grown inclusions of Chlamydia psittaci, strain ovine abortion (VR-656), as antigens (psittaci-ifat). Cervical IgG and IgA antibodies were detected by using fluorescein-conjugated anti-human IgA (secretory piece, Dako, Copenhagen, Denmark) in an LGV-IFAT at a 1:1 dilutiorl with material from specimens sent for chlamydial isolation. Enzyme immunoassay (EIA) was performed with a commercially available EIA kit (Chlamyset antibody EIA; Orion Diagnostica, Helsinki, Finland), which uses partially purified particles of C. trachomatis L2 as the antigen. In this assay, the sera were tested at a dilution of 1:100, and alkaline phosphatase-conjugated anti-human IgG conjugate was used. The antibody titer of each serum was calculated from the optical density by comparison with standard sera of known IFAT titers according to the instructions of the manufacturer. Statistical methods. Statistical analyses were performed with Student's t test and the x2 test. RESULTS Classification of the study population. During the acute PID episodes (3 to 6 years previously) 26 (38%) of 69 patients had had positive cervical or urethral cultures for C. trachomatis, and 20 (29%) of 68 patients had had positive cervical or urethral cultures for N. gonorrhoeae. Eight women had had both organisms isolated. In LGV-IFAT, nine patients (13%) had no serum IgG antibodies (titers, <1:16), 18 patients (26%) had low titers (1:16 to 1:64), and 43 patients (61%) had high titers (.1:128) (Fig. 1). The GMT of the positive IgG findings was 197. A CHLAMYDIAL ANTIBODIES AFTER PID 925 significant (.fourfold) change in antibody titers was demonstrated in 16 (36%) of 44 patients. Based on the microbiological and serologic findings, the women were classified into two groups, the Chlamydia (CT+) and non-chlamydia (CT-) groups. The CT+ group consisted of 51 women who had positive cultures for C. trachomatis, significant change in antichlamydial IgG antibody titer, high IgG (.1:128) antibody titer as demonstrated during the acute PID episode, or some combination of these indicators. The CT- group consisted of 19 women who had negative cultures for C. trachomatis and stable negative or low IgG antibody titers to C. trachomatis during the acute PID episode. Antichiamydial antibody findings during the follow-up examination. (i) Serum IgG antibodies. High titers (-1:128) were encountered in 22 women (32%), and low or negative titers were encountered in 48 women (68%) (Fig. 1). The GMT of the positive IgG titers was 73. Women belonging to the CT+ group significantly more often (21 of 51) had high (-1:128) IgG titers than women belonging to the CT- group (1 of 19; P < 0.01). Negative or low titers predominated in the CT- group (95%). By psittaci-ifat, 45 women (74%) had negative IgG titers (<1:8), and only 2 women (3%) had titers of -1:64. The GMT of the positive titers was 17. IgG antibodies were detected in the LGV-IFAT-positive cases belonging to the CT+ group only. Correlation between LGV-IFAT IgG antibodies and the Chlamyset antibody EIA IgG antibodies (Fig. 2) was determined from the follow-up sera. A fairly good correlation between these two tests was noted (r = 0.73). In general, the EIA seemed to give lower titers than LGV-IFAT. Negative findings (titers of < 1:16) were found by EIA in 40% (27 of 68) and by LGV-IFAT in 18% (12 of 68) of the tests. The GMT of the positive findings was 71 by EIA versus 73 by LGV- IFAT. The rate of high (.1:128) titers was 22% (15 of 68) by EIA and 32% (22 of 68) by LGV-IFAT. (ii) Serum IgA antibodies. IgA antibodies were demonstrated by LGV-IFAT in 33 (52%) of 63 women, 8 of whom had a titer exceeding 1:32. The GMT of the positive titers was 14. IgA antibody level in serum generally correlated EIA lgg titer '16. * so 0 so " a, a N & as.. " so r < I FAT IgG titer FIG. 2. Correlation of chlamydial IgG titers measured by LGV- IFAT and Chlamyset antibody EIA in 68 PID patients (follow-up samples).

3 926 PUOLAKKAINEN ET AL. with IgG antibody level in serum (r = 0.67) (Fig. 3). Thirty-one (63%) of 49 women in the CT+ group had serum IgA antibodies, compared with only 3 (18%) of 17 of those in the CT- group (P < 0.01). (iii) Cervical antibodies. Cervical IgG and IgA antibodies were found in 5 (11%) of 46 and 13 (28%) of 46 women, respectively. Cervical IgA antibodies were found in 10 (45%) of 22 women with high titers (.1:128) for IgG antibodies in serum and in 3 (7%) of 46 women with low or negative titers for IgG antibodies in serum (P < 0.001). Cervical IgA antibodies also correlated with the presence of serum IgA antibodies; i.e., 11 (85%) of 13 women with cervical IgA antibodies versus 16 (39%) of 41 women without cervical IgA antibodies had serum IgA antibodies (P < 0.01). Cervical IgG was less commonly found than cervical IgA, and it never occurred in the absence of cervical IgA. Positive IgA findings in cervical secretions were exclusively found in the CT+ group; 13 (29%) of 45 women in the CT+ group and none in the CT- group had detectable cervical IgA antibodies (P < 0.05). Persistence of LGV-IFAT IgG titers during the follow-up period. Duritng the follow-up period (3 to 6 years), 26 (43%) of the 60 women (whose sera were available for testing after exclusion of constantly seronegative patients) still had stable antibody titers, 26 (43%) showed a significant decrease in titers, and 8 (13%) showed a significant increase in titers. Forty-seven percent of women in the CT+ group and 27% of women in the CT- group showed a significant decrease in IgG titers. Stable titers were found in 43% of the CT+ group and 45% of the CT- group. Culture results. None of the women had positive cultures for C. trachomatis or N. gonorrhoeae during the follow-up examination. Historic and clinical findings. Nine patients (13%) gave a history of recurrent PID (six from the CT+ group and three from the CT- group), and six patients had a history of ectopic pregnancy (five from the CT+ group and one from the CT- group). Gynecologic examination revealed no abnormalities in 52 women (74%). Fourteen women (20%) showed visible cervical ectopy, two (3%) had mucopurulent endocervical discharge, and two (3%) had adnexal fullness on bimanual examination, suggesting pelvic adhesions. Women with cervical ectopy less frequently had serum IFAT IgG titer * *0 0 0 A 9 0@ 00 as a * * ' 0 r-0.67 < IgAter FIG. 3. Correlation of chlamydial serum IgA and IgG antibodies in 63 PID patients (follow-up samples). TABLE 1. Fertility status in the CT+ and CT- groups No. (%) of women J. CLIN. MICROBIOL. Group" Fertile' Childlessc Infertile CT+ 17 (33) 23 (45) 13 (25) CT- 8 (42) 9 (47) 3 (16) afor an explanation of groups, see Materials and Methods. Three women belonged to both groups (two in the CT+ group and one in the CT- group). bhistory of intrauterine pregnancy after the PID episode. Voluntarily or by use of contraceptives. LGV-IFAT IgG antibodies (6 of 12 versus 50 of 56; P < 0.01) on the follow-up visit than those without ectopy, but the GMT of positive sera was higher (203 versus 65). In cervicovaginal Pap smears, Doderlein flora (lactobacilli) was present in 28 women (40%), and coccoidal or mixed bacterial flora dominated in 42 smears (60%). In addition, four women had metaplastic atypia, and none had atypia consistent with dysplasia. Clue cells suggesting bacterial vaginosis were noted in 17 women (24%). Infertility after PID. Distribution of the women in the CT+ and CT- groups according to their fertility status is shown in Table 1. The involuntarily childless women belonged more often to the CT+ group, but the difference was not statistically significant. Twenty-five women (36%) had an intrauterine pregnancy (tuboplasty had been performed in one case). Of these women, 16 delivered, 2 had spontaneous abortions and 11 underwent termination of pregnancy. In addition, two of these women also had an extrauterine pregnancy. Forty-five women (64%) were childless during the follow-up period, either voluntarily (32 women, of whom 19 were using contraceptives) or involuntarily (13 women). In addition, three women had an intrauterine pregnancy but suffered later from fertility problems. Three women had undergone hysterectomy, and two had undergone tubal sterilization. At the time of investigation, 14 women were trying to get pregnant. Seven women had infertility of short duration (<1 year) or had a history of spontaneous abortion. Five of the remaining seven women had undergone diagnostic laparoscopy and hysterosalpingography. Tubal occlusion was noted in three women, two of whom had subsequently been operated on. Findings on IgG and IgA in serum and local IgA antibody for these patient groups are presented in Table 2. There were no differences in the prevalence or GMT of serum antibodies between the different subgroups. However, cervical IgA antibodies were less prevalent among the involuntarily childless women in the CT+ group (statistically not significant). Correlations between the lower-genital-tract culture results for N. gonorrhoeae and C. trachomatis during the index PID episode and subsequent fertility status are shown in Table 3. No significant differences were found between the groups, indicating that lower-genital-tract cultures are poor in predicting possible tubal damage. DISCUSSION Several different tests, including the complement fixation (CF) test, various immunofluorescence tests (micro-ifat, inclusion IFAT), and EIAs, are currently used to determine chlamydial antibodies. These tests probably measure antibodies against different antigenic determinants of Chlamydia species, depending on the antigen used. The CF test with the group-specific chlamydial antigen mainly detects antibody

4 VOL. 23, 1986 TABLE 2. Group Serum IgG and IgA and local IgA antibody findings in the CT + and CT- groups No. (%) of women with: Serum IgGa Serum IgAb Cervical IgA CT + Fertile 14 (88) 8 (53) 7 (44) Voluntarily 20 (91) 14 (67) 5 (26) childless Infertile 13 (100) 9 (69) 1 (10) CT- Fertile 4 (50) 1 (14) 0 Voluntarily 7 (78) 2 (25) 0 childless Infertile a Titer, -1:16. GMTs for the groups were as follows: CT+ fertile, 91; CT+ involuntarily childless, 79; CT+ infertile, 93; CT- fertile, 23; and CTvoluntarily childless, 39. btiter,.1:8. responses elicited in C. psittaci infections and in complicated C. trachomatis infections only (except LGV). Tests based on immunofluorescence technique are more sensitive than the CF test but have the disadvantage of being subjective, rendering direct comparison of results between laboratories (and interpreters) difficult. Good correlation between the micro-immunofluorescence test and LGV-IFAT, however, has been noted (Saikku, unpublished observations). Objective tests by the EIA principle are to be introduced in chlamydial serology, but they are not entirely acceptable until carefully evaluated. In the Finnish female blood donor population, a chlamydial IgG antibody titer.1:128 (by LGV-IFAT) was found in 4.1%, and titers.1:256 were found in 1.6% (Puolakkainen, unpublished observations), indicating that such high IgG titers are seldom encountered in a normal female population and obviously are of significance, representing current or past chlamydial infection. However, as persistence of chiamydial antibodies after acute infection is a poorly characterized phenomenon, definitions of so-called high single IgG antibody titers with clinical significance (4) should be interpreted with caution. Chlamydial CF antibody titers may disappear within a few months, although persistence at practically the same level for a long time (up to 8 years) has been noted (5). According to Matthiesen and Volkert (7), CF antibodies decrease after the infection, first rapidly and then more slowly. Persistence of chlamydial antibodies detectable by immunofluorescence tests after genital infection due to C. trachomatis is poorly documented. According to Mardh (6), the initially high IgG titers disappeared within 1.5 years in some tetracyclinetreated patients, but much longer persistence of antibodies has been suggested (9, 22). IgM antibodies, associated with acute disease, are shown to persist for 8 to 10 weeks after genital infections (24), but may persist for even longer periods (17). Regardless of the initial titer, 43% of the patients with chlamydial PID showed stable IgG titers (by LGV-IFAT) for up to 6.3 years in our study, although only 13% of women were registered to have recurrences. Asymptomatic recurrences, however, cannot be excluded. We also found antibodies detectable by our psittaci-ifat (the test is obviously group specific, because the ovine abortion strain of C. psittaci we used is not encountered in Finland) infrequently (26%), while those measured by LGV-IFAT were prevalent CHLAMYDIAL ANTIBODIES AFTER PID 927 (83%). These results might indicate that antibodies against chlamydial lipopolysaccharide (group-specific antibodies) disappear more rapidly than antibodies against chlamydial protein(s) (species-specific antibodies), which thus tend to persist. This possibility is in accordance with previous findings of CF (group-specific) antibodies (5, 7). The significance of serum and local IgA antibodies in chlamydial infections has remained unclear. Short-lived serum IgA antibodies have been proposed as a marker for active C. trachomatis infection, especially in nongonococcal urethritis patients (3). Local IgA antibodies have also been considered a sign of active ongoing infection (8), and the presence of cervical IgA and IgG antibodies has been shown to correlate with the isolation of C. trachomatis (23). However, opposite findings have been reported, too. The correlation between local antibodies and the isolation of C. trachomatis has been shown to be much weaker than that between serum antibodies and local antibodies (14). In addition, the persistence of urethral IgA antibodies for at least 3 years (21) and a high background level of local antibodies in a venereal-disease clinic population (18) diminish the diagnostic value of local antibodies as an indicator of acute infection. Brunham et al. (2) have demonstrated that secretory IgA antibodies in cervical secretions correlate inversely with the quantitative recovery of C. trachomatis from the cervix, suggesting an inhibitory effect of cervical antibodies on the isolation of C. trachomatis. The high prevalence of local antibodies in our material may be an explanation for the failure to isolate C. trachomatis in all cases, although a carrier rate of 5% has been found in this same department (12). The samples were processed in the same laboratory under identical conditions during these years. The only modification was a change from irradiated cells to cycloheximide-treated cells, which should increase the sensitivity (15). We cannot, however, exclude the possibility of active upper genital infection in some women with negative cervical cultures in the presence of local antibodies. PID is one of the major causes of infertility in women. After one, two, and three or more episodes, the frequency of post-pid infertility is 11, 23, and 54%, respectively (27). In our material, 16 women (42%) were involuntarily childless. This figure is quite high compared with those reported by Westrom (27) and knowing that repeat PID episodes were infrequent in our study group. One explanation is that severer cases were overrepresented in the present material, since all patients had been hospitalized because of severe symptoms. Severity of the inflammatory tubal changes significantly correlates with subsequent infertility (27). Five of twelve involuntarily childless women in the CT+ group were also culture positive for N. gonorrhoeae in the acute phase. This fact probably contributes to infertility (26). Increased risk for an ectopic pregnancy is another poten- TABLE 3. Correlation between lower-genital-tract culture results during the index PID episode and subsequent fertility status during the follow-up C. trachomatis or No. (%) of womena N. gonorrhoeae or both isolated from the Fertile Infertile lower genital tract Yes 14 (67) 7 (33) No 8 (57) 6 (43) avoluntarily childless women and three women belonging to both groups were excluded from these calculations.

5 928 PUOLAKKAINEN ET AL. tial sequela of PID. In a Swedish report (27), a 7- to 10-fold risk for an ectopic pregnancy after PID has been noted. In our small group, six patients wanting children had an ectopic pregnancy compared with 25 who had an intrauterine pregnancy, giving a ratio of 1:4, which is considerably greater than that observed in the Swedish study (27). Our results confirm the previous studies, suggesting that a history of PID is the most important risk factor for an ectopic pregnancy. An interesting difference was noted between the infertile women and those with intrauterine pregnancy in the CT+ group: infertile women had cervical IgA antibodies less often than those with intrauterine pregnancy (10 versus 44%), suggesting that the presence of local antichlamydial IgA antibodies in genital secretions might protect tissue from considerable damage, but due to small numbers, the difference was not satistically significant. It is possible that in infertile women, the production of local IgA is deficient, or if the antibody is produced, that it is consumed or destroyed (1). The protectiie role of local antibodies in genital secretions deserves further study. ACKNOWLEDGMENTS We thank Marja-Liisa Kauppinen and Hella Sarjakivi for technical assistance. This study was supported by grants from the Finnish Cultural Foundation and from Orion Diagnostica, Espoo, Finland. J. CLIN. MICROBIOL. LITERATURE CITED 1. Blake, M., K. K. Holmes, and J. Swanson Studies on gonococcus infection. XVII. IgAl-cleaving protease in vaginal washings from women with gonorrhoea. J. Infect. Dis. 139: Brunham, R. C., C.-C. Kuo, L. Cles, and K. K. Holmes Correlation of host immune response with quantitative recovery of Chlamydia trachomatis from the human endocervix. Infect. Immun. 39: Cevenini, R., I. Sarov, F. Rumpianesi, M. Donati, C. Melega, C. Varotti, and M. 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