Persistence of chlamydial antibodies after pelvic inflammatory disease

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0095-1137/86/050924-05$02.00/0

Persistence of Chlamydial Antibodies

after

Pelvic

Inflammatory Disease

MIRJA PUOLAKKAINEN,l* ERVO VESTERINEN,2 ESKO PUROLA,2 PEKKASAIKKU,1 ANDJORMAPAAVONEN3t

Department ofVirology, University ofHelsinki,' andDepartments IandII ofObstetrics and Gynecology, University CentralHospital,2 Helsinki, andDepartment of Clinical Sciences, University of

Tampere,'

Tampere, Finland

Received 8 November 1985/Accepted 29 January 1986

The persistence of chlamydial immunoglobulin G (IgG) antibodies and long-term sequelae of pelvic inflammatory disease(PID)werestudied in70womenwhohad been treated for PID 3to6yearspreviously.

Fifty-one women had had PID associated with Chlamydia trachomatis infection (Chlamydia group), and 19 womenhad hadPIDnotassociated with C.trachomatis(non-Chlamydiagroup).Chlamydial IgGantibodies,as

determinedby the indirectimmunofluorescencetestwithinclusions of C. trachomatisL2asantigens,persisted

atstable levelsin 43% ofthewomenforupto6years;43% ofthewomenshowedadecreasein IgGtiter, and

13% showedanincrease.IgAantibody levelsinserumcorrelated withIgG antibodylevelsinserumandwith

thepresence of cervical IgAantibodies. Bothserum antibodiesandcervical IgA antibodies were moreoften

found in the Chlamydia group. Forty-two percent of the women were infertile. Every fifth subsequent pregnancy wasectopic. ThepresenceofcervicalIgAantibodies mightprotectthewomenfromtubaldamage.

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). Inisolation positivemale

ure-thritis 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 re-sponse. The geometric mean titer (GMT) by the indirect immunofluorescence antibody test (IFAT) for

immunoglob-ulin G (IgG) in urethritis is low, but moderately elevated GMTs are observed among patients with cervicitis, and

clearly elevated titers are seenamongpatients with compli-catedgenital infections(25). Sincetheearly disease maybe

asymptomatic, antibodies have already appeared in many cases by the time serological diagnosis is attempted,

ham-peringthedemonstration of seroconversions.Thehigh

back-groundprevalence ofantibody, especially in sexually

trans-mitted disease clinic populations, also hampers the

di-agnostic utility of seroconversion. In addition, chlamydial antibodies seem to persist even after the treatment of the

infection, making assessment ofa single IgGantibody titer difficult. The diagnosis ofgenitalchlamydial infections can-not be based on measurement ofspecific 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 under-taken 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,

long-termsequelaeof PIDwereanalyzed, especiallyinrelationto pastand present serologicalresults.

*Correspondingauthor.

tPresent address: Department of Obstetrics and Gynecology, School ofMedicine, UniversityofWashington, Seattle, WA98104.

MATERIALS AND METHODS

Study population. In 1983, an invitation letter for free gynecologicexaminationwassentto 98 women whohad been treatedfor PID with appropriate antibiotics at the I and II Departments of Obstetrics and Gynecology, Helsinki University Central Hospital, between 1977 and 1980. The clinical diagnosis of PID was based on clinical criteria

includinghistoryof lower abdominalpain of less than 3-week

duration; abnormal vaginal discharge; adnexal tenderness,

usually withapalpable mass;erythrocytesedimentationrate of >15mm/h; and fever of>38°C (11). Cervicaland urethral cultures for C. trachomatis andNeisseriagonorrhoeaewere performed as previously described

(12).

Acute- and

conva-lescent-phasesera wereobtained,andserumIgG antibodies

toC.trachomatisweredeterminedasdescribed elsewhere in detail (16).

Seventy women (71%) responded to the letter and were examined attheoutpatient clinic ofthesame departmentin 1983 and 1984. Informed consent was obtained from all

patients. Themeanageof thewomen was31.5 years(range,

20 to48 years), and the mean follow-up time afterthe PID

episode was 5.0years (range, 3.1to 6.3years).

Hospital records from the previous PID episodes were

carefully reviewed,withspecial emphasisonmicrobiological 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

prac-tices, fertilityproblems,andothergenitalsymptoms. 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 endocervixwerestainedby

Papanicolaou's original

method.

Specimens from the cervix and urethra were obtained for isolation ofC. trachomatis and N. gonorrhoeae.

Microbiological methods.

Cycloheximide-treated McCoy

cells were used for the isolation of C.

trachomatis,

and 924

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70

*

follow up

60

50-40

30

20-10

<16

16-64

>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-upexamination.

growth was detected byiodine

staining

oftheinclusions(15).

Isolation of N. gonorrhoeae was performed by routine

methods

(10).

AnIFAT wascarriedoutwith 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 dilu-tions,andfluorescein-conjugated anti-human IgG(Wellcome ResearchLaboratories, Beckenham, England)andIgA

(Kal-lestad Laboratories, Inc., Chaska,

Minn.)

were used to detect the antigen-bound antibodies. In LGV-IFAT, IgG titers.1:128wereconsidered high(4). Serumsamples were

also tested forthepresenceofIgG antibodies byIFAT 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

com-mercially availableEIAkit(Chlamyset antibodyEIA;Orion Diagnostica, Helsinki, Finland), whichusespartially purified

particles

ofC. trachomatis L2astheantigen. In this assay, the sera were tested at a dilution of 1:100, and alkaline phosphatase-conjugated anti-human IgG conjugate was

used. Theantibody titer ofeach serum wascalculatedfrom

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 thex2 test.

RESULTS

Classification of the studypopulation.DuringtheacutePID

episodes(3to6yearspreviously)26(38%)of69patients had hadpositivecervical or urethral cultures for C. trachomatis, and 20 (29%) of68 patients

had

had positive cervical or urethralcultures for N. gonorrhoeae. Eight women had had

bothorganisms isolated.

In LGV-IFAT, nine patients (13%) had no serum IgG

antibodies (titers, <1:16), 18 patients (26%) had low titers

(1:16to 1:64), and 43patients (61%) had high titers

(.1:128)

(Fig. 1). The GMT of the positive IgG findings was 197. A

significant (.fourfold) changeinantibody titerswas demon-strated 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

anti-bodytiter, high IgG(.1:128) antibody titerasdemonstrated

duringtheacutePIDepisode,or somecombinationof these

indicators. The CT-groupconsisted of19womenwhohad negative cultures for C. trachomatis and stable negative or lowIgG antibody

titers

to C. trachomatis during the acute PIDepisode.

Antichiamydial antibody findings during the follow-up ex-amination. (i) Serum IgG antibodies. High titers (-1:128)

wereencountered in 22 women (32%), and low or negative titers were encountered in 48 women (68%) (Fig. 1). The

GMT ofthepositive IgG titerswas73. Womenbelongingto theCT+ group significantly more often (21 of51) hadhigh

(-1:128)IgGtiters than womenbelongingtothe CT- group (1of 19;P < 0.01). Negative orlowtiters predominated in theCT- group(95%).

Bypsittaci-IFAT,45 women(74%)hadnegative IgGtiters (<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+ grouponly.

Correlation between LGV-IFAT IgG antibodies and the

Chlamysetantibody EIA IgG antibodies (Fig. 2) was

deter-mined from the follow-up sera. A fairly good correlation

between thesetwo tests wasnoted (r= 0.73). Ingeneral,the EIA seemedto give lower titersthanLGV-IFAT. Negative findings (titers of<1:16)werefoundbyEIA in40% (27 of 68)

andby LGV-IFAT in 18% (12 of68) of thetests. The GMT

ofthe positive findings was 71 by EIA versus 73 by LGV-IFAT.Therateof high

(.1:128)

titerswas22%(15of68) by EIAand 32% (22 of 68) by LGV-IFAT.

(ii) Serum IgA antibodies. IgA antibodies were demon-stratedby LGV-IFAT in 33(52%) of63 women, 8 ofwhom had a titer exceeding 1:32. The GMT of the positive titers was 14. IgA antibody level in serum generally correlated

EIA

lgG

titer

2048

-1024

512

256

128

64

32

16

'16

@0

.

A

so

* so 0 so

" a, a

N

&

as

..

"

<16 16 32 64

r-0.73

128 512

256 1024

IgG titer

IFAT

FIG. 2. Correlationofchlamydial IgG titers measured by LGV-IFAT and Chlamyset antibody EIAin 68 PID patients (follow-up samples).

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with IgG antibody level in serum (r = 0.67) (Fig. 3).

Thirty-one (63%) of 49womenin the CT+ grouphadserum

IgAantibodies, compared with only3(18%) of 17 of those in theCT- group (P< 0.01).

(iii) Cervical antibodies. Cervical IgG and IgA antibodies

were found in 5 (11%) of 46 and 13 (28%) of46 women,

respectively. Cervical IgA antibodieswerefound in 10 (45%)

of 22women with high titers (.1:128) for IgG antibodies in

serumandin 3 (7%) of 46women with lowornegative titers

for IgG antibodies in serum (P < 0.001). Cervical IgA antibodies also correlated with the presence ofserum IgA

antibodies; i.e., 11 (85%) of 13 women with cervical IgA

antibodiesversus16(39%) of 41womenwithoutcervical IgA

antibodies had serum IgA antibodies (P < 0.01). Cervical IgGwaslesscommonly found thancervical IgA, and itnever

occurredin the absence of cervical IgA.

Positive IgA findings in cervical secretions were

exclu-sivelyfound in the CT+ group;13(29%) of 45womenin 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 to6 years), 26 (43%) of the 60women (whoseserawereavailable fortesting after exclusionofconstantly 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-sevenpercentofwomenin the CT+ groupand 27% of

women in the CT- group showed a significant decrease in

IgG titers. Stable titerswerefoundin43% oftheCT+group

and45% oftheCT- group.

Cultureresults. Noneofthe womenhadpositive cultures

for C. trachomatis orN. gonorrhoeae during the follow-up examination.

Historicand clinical findings. Nine patients (13%)gave a

history ofrecurrent PID(six from the CT+ groupand three

from the CT- group), and six patients had a history of

ectopic pregnancy (five from the CT+ group andone from the CT- group). Gynecologic examination revealed no

abnormalities in 52 women (74%). Fourteen women (20%) showedvisiblecervicalectopy, two (3%)hadmucopurulent endocervical discharge, andtwo (3%) hadadnexal fullness on bimanual examination, suggesting pelvic adhesions.

Women with cervical ectopy less frequently had serum

IFAT

IgG

titer

1024

1

0

*

512

256 128 64 32

16

416

@0 0 0

0 *0 0 0

A 9

0@ 00

as a

*

_ _'

0

r-0.67

<8 8 16 32 64

IgAter

FIG. 3. Correlation ofchlamydialserumIgAandIgGantibodies in63 PIDpatients (follow-up samples).

TABLE 1. Fertility status in the CT+ and CT- groups

No.(%) ofwomen

Group" Fertile' Childlessc Infertile

CT+ 17(33) 23 (45) 13 (25)

CT- 8(42) 9(47) 3(16)

aFor anexplanation ofgroups, see Materials and Methods. Three women

belongedtobothgroups (twointheCT+group and oneintheCT-group).

bHistory of intrauterinepregnancyafterthe PID episode.

Voluntarilyorbyuseof contraceptives.

LGV-IFATIgG antibodies(6of 12 versus 50 of 56; P<0.01) on the follow-up visit than those without ectopy, but the GMTof positive serawas higher (203 versus 65).

In cervicovaginal Pap smears, Doderlein flora (lac-tobacilli)was present in 28 women(40%), and coccoidal or

mixed bacterial flora dominated in 42 smears (60%). In

addition, four women hadmetaplastic atypia, and nonehad atypia consistent with dysplasia. Clue cellssuggesting bac-terialvaginosis were noted in 17 women (24%).

Infertility after PID.Distribution ofthe womenin the CT+ and CT- groupsaccordingtotheirfertility status is shown in

Table 1. The involuntarily childless women belonged more

often tothe CT+group, but the difference was not statisti-cally significant.

Twenty-five women (36%) hadan 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 womenalso had an extrauterine pregnancy.

Forty-five women (64%) were childless during the fol-low-up period, either voluntarily (32 women, of whom 19 wereusingcontraceptives) orinvoluntarily (13women). 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.

Atthe timeofinvestigation, 14 women weretryingtoget pregnant.Sevenwomenhadinfertility ofshortduration(<1

year)or had ahistory ofspontaneous abortion. Fiveofthe

remainingseven women had undergone diagnostic laparos-copyandhysterosalpingography. Tubalocclusionwasnoted in three women, two of whom had subsequently been

operatedon.

FindingsonIgG and IgAin serumandlocalIgAantibody forthese patient groupsarepresentedin Table 2. Therewere nodifferences in theprevalenceorGMT ofserumantibodies

between the different subgroups. However, cervical IgA

antibodieswerelessprevalentamongthe

involuntarily

child-less womenin the CT+ group

(statistically

not

significant).

Correlations between the

lower-genital-tract

culture re-sults for N. gonorrhoeae and C. trachomatis

during

the

indexPIDepisodeandsubsequentfertilitystatusareshown in Table 3. No significant differences were found between the groups, indicating that

lower-genital-tract

cultures are poorin predicting

possible

tubaldamage.

DISCUSSION

Several different tests,

including

thecomplement fixation (CF) test, various immunofluorescence tests

(micro-IFAT,

inclusionIFAT),andEIAs, are

currently

usedtodetermine chlamydial antibodies. These tests

probably

measure

anti-bodiesagainstdifferent

antigenic

determinantsof

Chlamydia

species,dependingonthe

antigen

used. TheCFtestwith the

group-specific chlamydial antigen mainly detects

antibody

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TABLE 2. SerumIgGand IgA and localIgA antibody findings in theCT+ and CT-groups

No.(%) ofwomenwith: Group

SerumIgGa SerumIgAb CervicalIgA 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 0 0 0

aTiter,-1:16.GMTs forthe groups were asfollows: CT+fertile,91; CT+

involuntarily childless, 79; CT+ infertile, 93; CT- fertile, 23; and CT-voluntarily childless,39.

bTiter,.1:8.

responses elicited in C. psittaci infections and in compli-cated C. trachomatis infections only (except LGV). Tests basedon immunofluorescence techniqueare moresensitive

than the CFtestbut have thedisadvantage of being subjec-tive, rendering directcomparison ofresultsbetween labora-tories (and interpreters) difficult. Good correlationbetween the micro-immunofluorescence test and LGV-IFAT,

how-ever, has been noted (Saikku, unpublished observations). Objectivetestsby the EIAprinciple are tobe introducedin

chlamydial serology, but they are not entirely acceptable

until carefully evaluated.

IntheFinnish female blood donorpopulation,a chlamyd-ial IgG antibody titer .1:128 (by LGV-IFAT)was found in

4.1%, andtiters

.1:256

werefound in 1.6%(Puolakkainen, unpublished observations), indicating that such high IgG titersareseldom encounteredinanormal femalepopulation and

obviously

are of

significance, representing

current or pastchlamydial infection. However, as

persistence

of chia-mydial antibodies afteracuteinfectionisapoorly character-ized phenomenon, definitions of so-called high single IgG antibodytiters withclinical significance (4) should be

inter-preted with caution.

ChlamydialCFantibodytiters maydisappearwithinafew months, although persistence at practically the same level

foralong time(upto8years) has beennoted(5). According

toMatthiesenandVolkert(7), CF antibodiesdecrease after

theinfection, first rapidlyand then moreslowly. Persistence ofchlamydial antibodies detectable by immunofluorescence

tests aftergenital infection dueto C. trachomatis is poorly

documented. According toMardh (6), the initiallyhigh IgG

titers disappeared within 1.5 years in some

tetracycline-treated patients, but much longer persistence of antibodies

has been suggested (9, 22). IgMantibodies, 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% ofthe patients with

chlamydialPID showed stableIgGtiters (by LGV-IFAT) for up to 6.3 years in our study, although only 13% of women were registered to have recurrences. Asymptomatic recur-rences, however, cannot be excluded. We also found anti-bodies detectable by our psittaci-IFAT (the test is obviously group specific, because the ovine abortion strain of C. psittaciwe usedisnotencountered inFinland) infrequently

(26%),

while those measured by LGV-IFAT were prevalent

(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

findingsof CF(group-specific) antibodies (5, 7).

The significance of serum and local IgA antibodies in

chlamydialinfections has remained unclear. Short-lived se-rum IgA antibodies have been proposed as a marker for

activeC. trachomatisinfection, especiallyinnongonococcal

urethritis patients(3). Local IgAantibodies have also been

considered a sign of active ongoing infection (8), and the presenceof cervicalIgAandIgGantibodies has been shown to correlate with the isolation of C. trachomatis(23). How-ever, opposite findings have beenreported, too. The

corre-lation 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 clinicpopulation (18) dimin-ish thediagnosticvalueoflocalantibodiesas anindicator of

acute infection.

Brunham etal. (2)have demonstrated that secretory IgA antibodiesincervical secretionscorrelateinverselywith the

quantitative recovery of C. trachomatis from the

cervix,

suggesting an inhibitory effect of cervical antibodieson 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 of5% has been found in this same department

(12). The samples were processed in the same laboratory

under identical conditions during these years. The only modificationwas achange from irradiated cellsto cyclohex-imide-treated cells, which should increase the sensitivity

(15). We cannot, however, exclude thepossibility of active

upper genital infection in some women with negative cervical cultures inthepresence of local antibodies.

PID is one ofthe

major

causes of

infertility

in women.

Afterone, two, and threeor more

episodes,

the

frequency

of post-PID

infertility

is11, 23,and

54%, respectively

(27). In our

material,

16women

(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 severercases wereoverrepresented in the presentmaterial, since all

patients

had been

hospitalized

because ofsevere symptoms.

Severity

ofthe

inflammatory

tubal changes

sig-nificantly correlateswith subsequent infertility(27). Fiveof

twelveinvoluntarily childlesswomenintheCT+group were alsoculture

positive

forN. gonorrhoeaeintheacutephase.

Thisfactprobably contributes to infertility(26).

Increasedrisk foranectopicpregnancy is another poten-TABLE 3. Correlation betweenlower-genital-tract culture results

duringtheindexPIDepisodeandsubsequentfertility status duringthefollow-up

C.trachomatisor No.(%)of womena N.gonorrhoeaeor

bothisolated fromthe Fertile Infertile lowergenitaltract

Yes 14(67) 7(33)

No 8 (57) 6(43)

aVoluntarilychildless women and three womenbelongingtoboth groups wereexcluded from thesecalculations.

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tial sequela of PID. In a Swedish report (27), a 7- to10-fold risk for an ectopic pregnancy after PID has been noted. In oursmall group, six patients wanting children had an ectopic pregnancy compared with 25 who had an intrauterine

preg-nancy, 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 isthe 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 antibodiesless 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

differ-ence was not satistically significant. It is possible that in

infertilewomen, 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

secre-tions deserves further study.

ACKNOWLEDGMENTS

We thankMarja-LiisaKauppinen andHella Sarjakivifor technical assistance.

This study was supported by grants from the Finnish Cultural Foundation andfrom Orion Diagnostica, Espoo, Finland.

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