Diagnosis and prevalence of persistent chlamydia infection in infertile women: tissue culture, direct antigen detection, and serology*t

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1 FERTILITY AND STERILITY Copyright" 1991 The American Fertility Society Vol. 55, No.2, Fehruary 1991 Printed on acid free paper in U.S.A. Diagnosis and prevalence of persistent chlamydia infection in infertile women: tissue culture, direct antigen detection, and serology*t Henning Thejls, M.D.:!: Judy Gnarpe, Ph.D. Orjan Lundkvist, M.D. II Gun Heimer, M.D. II Gregor Larsson, M.D. II Arne Victor, M.D. II Gavle Central Hospital, Gavle, and Institution of Obstetrics and Gynecology, Akademiska Sjukhuset, Uppsala University, Uppsala, Sweden Specimens for chlamydial culture, direct fluorescent antibody (DF A) test, two enzyme immunoassays (EIA) for antigen detection, and serum for chlamydial antibodies were collected from 256 infertile women. Specimens were taken from the tubes during tuboplasty and from the cervix and endometrium during laparoscopy or tuboplasty. Antibodies to Chlamydia trachomatis were found four times more often in patients with signs of prior pelvic inflammatory disease (PID) than in infertile women with normal pelvic findings. Only 48 (37%) of 131 patients with signs of prior PID had a history of PID. Ten or more C. trachomatis elementary bodies (EBs) per smear were found in 21 (8.2%) of 256 patients. Six patients had a positive culture or a positive antigen EIA test. All six had high numbers of EBs in the DF A test. We conclude that routine culture and EIA antigen tests detect only a minority of persistent chlamydia infections in this population, but subjective factors in the interpretation ofdfa methods must be considered. Fertil Steril55:304, 1991 The role of Chlamydia trachomatis as an etiological factor in female infertility is well recognized. 1,2 Whether and to what extent chlamydia persists as a clinically silent infection in some infertile women has been a matter of debate, and studies have given divergent results. Chlamydia has been cultured from the fallopian tubes, endometrium, or peritoneal fluid of asymptomatic infertile women by some workers. 3 -s Other workers have failed to demonstrate chlamydia by culture in this popula- Received April 20, 1990; revised and accepted September 20, * Supported by Gavleborgs Lans Landsting, Gavle, Sweden and Pfizer AB, Taby, Sweden. t Presented in part at the XIII W orid Congress on Fertility and Sterility, Marrakesh, October 1 to :j: Reprint requests: Henning Thejls, M.D., Department of Obstetrics and Gynecology, Gavle Central Hospital, Gavle, Sweden. Department of Clinical Bacteriology, Gavle Central Hospital. II Institution of Obstetrics and Gynecology, Akademiska Sjukhuset, Uppsala University. tion,7-11 and Gump et al. 12 reported only one positive endometrial culture in 185 specimens. Clinical investigations using sequential swabs from the same location and/or multiple blind passages in tissue culture have shown that a standard culture procedure with one swab and reading the tissue cultures after primary inoculation can have sensitivity of <70% when tested against positive results of any swab or passage number.13,14 The sensitivity for specimens taken from the endometrium and the fallopian tubes seems to be lower than for cervical specimens. 15 Inhibition of chlamydia by host defense factors is one possible explanation why chlamydia may fail to grow in tissue culture in spite of its presence in the specimen. Methods for direct antigen detection with monoclonal antibodies are independent of the viability of chlamydia in the specimen sampling. Use of these tests may therefore theoretically compensate for culture failures caused by inhibited or neutralized chlamydial elementary bodies (EBs). The aim of this study was to evaluate the preva- 304 Thejls et al. Persistent chlamydia and infertility

2 lence of silent chlamydia infection in a population of infertile women using direct antigen tests, serology, and culture and to relate it to history and clinical findings. MATERIALS AND METHODS Women from two different clinics undergoing evaluation for infertility were enrolled in the study when they were admitted for laparoscopy or tubal surgery. A total of 256 infertile women were included in the study over a period of 18 months. Case histories and evaluation of pelvic pathology were recorded for all patients. Tubal specimens were collected from 53 cases; in 5 of these patients tests from the cervix and endometrium had been taken a few months earlier during laparoscopy, and in 1 case cervical and endometrial specimens were missing. The 2 M sucrose-phosphate medium with the specimens from the endometrium from 4 patients had leaked so culture could not be done. A history was taken concerning prior pelvic inflammatory disease (PID), prior chlamydial infection, pregnancies, pelvic operations, chronic symptoms of discharge, pelvic pain, and metrorrhagia. Serum was collected from all patients for antibody titration using both enzyme immunoassay (EIA) and microimmunofluorescence (MIF) techniques. A group of 1,078 consecutive pregnant women who were investigated during the same period was used for comparison of serum antibody prevalence. Serum was collected in the first trimester of pregnancy. Specimen Collection Cervix At operation, the cervix was first cleansed with a large dacron swab. Endocervical specimens were then collected for the chlamydiazyme test (Abbott Laboratories, North Chicago, IL) using the two small cotton swabs provided with the test kit. The first was used for another cleansing of the endocervix and then discarded according to the manufacturers' instructions. The other was rotated a few seconds in the endocervix and placed in the medium after ensuring that the small drop of medium was in the bottom of the tube. Specimens for direct antigen EIA test (Pharmacia AB, Uppsala, Sweden) were then collected from the cervix using the test kit according to the manufacturer's instructions. Specimens for culture were taken from ectocervix and endocervix with a C. trachomatis alginate swab (BioHospital AB, Stockholm, Sweden), shown to be atoxic to chlamydia, and placed in 2 M sucrose-phosphate chlamydia transport medium. Another swab was taken in the same manner and squeezed onto a MicroTrak (Syva Co., Palo Alta, CA) slide covering the 8-mm well evenly. The slide was air dried and fixed with acetone. Endometrium An instrument for collecting endometrial biopsies (Gynoscan, AKZO, Organon AB, V. Frolunda, Sweden) was introduced into the uterine cavity after retracting the instrument into its cover to minimize contamination from the cervix. Some of the material from the endometrial biopsy was placed in 2 M sucrose-phosphate transport medium to be cultured and another part of the biopsy was pressed onto a MicroTrak slide using a swab as described above. Fallopian Tubes Small biopsies or swab-specimens from the fimbriated end of the tube were taken during tuboplasty to be examined by MicroTrak and culture. Laparoscopic Evaluation Patients were classified into three different groups according to the findings at operation. Prior PID-patients having hydrosalpinx with or without peritubal adhesions, and patients with fimosis of the fimbriated end of the tubes of any degree, peritubal and periovarial adhesions when no other cause of the adhesions could be suspected from history or clinical findings; patients with adhesions caused by prior pelvic operations, endometriosis, or of uncertain origin; and patients with normal pelvic findings. Laboratory Procedures All laboratory procedures were performed at the Department of Bacteriology at the Gavle Central Hospital. All specimens taken at the clinic in Uppsala, approximately 100 km from Gavle, were sent to the laboratory by express mail on the day of collection. They were properly refrigerated before and during transportation, or frozen at -70 C if taken on a Friday and sent the following Monday. Serum samples were separated from cells before transportation to the laboratory. Vol. 55, No.2, February 1991 ThejIs et al. Persistent chlamydia and infertility 305

3 Serological Tests Microimmunofiuorescence Technique. Indirect MIF for determination of serum immunoglobulin (Ig)G and IgM antibodies to chlamydia was performed according to Treharne et al. 16 on slides obtained from the Institute of Ophthalmology, University of London, London. Egg-cultured preparations of C. trachomatis serovars D-K and C. pneumoniae (strain IOL-207) were used as antigens and normal yolk sac for a negative control. Sera were screened at an initial titer of 1:64 for IgG and 1:16 for IgM. Positive specimens were repeated and titrated to the end point. A fluorescein isothiocyanate labeled antihuman IgG produced in sheep (Wellcome Foundation Ltd., Dartford, England) and antihuman IgM produced in rabbit (DAKO PATTS a/s, Glostrup, Denmark) were used. Chlamydia EIA Technique. All serum samples were tested for IgG and IgM antibodies using an EIA technique. Partially purified preparations of elementary bodies obtained from McCoy cell cultures of C. trachomatis L2 were used to coat microtiter plates. Serum was diluted 1:100 and 100 ~L of diluted sample added to each well. After incubation of the sealed plates at 37 C for 2 hours, a thorough washing with phosphate buffered saline-tween (0.02%) was done. Swine antihuman IgG and IgM (Orion Diagnostica, Espoo, Finland) were added in appropriate dilutions and sealed plates left to incubate for 16 to 20 hours at room temperature. After thorough washing procedures, plates were incubated at 3TC with substrate solution (p-nitrophenol phosphate in 1 M diethanolamine buffer) and reactions stopped by addition of 1 N NaOH after 30 minutes. Absorbances were read at 490 nm in a scanning spectrophotometer. Standards prepared by pooling serum with appropriate titers in MIF were run with each plate. When IgM antibodies were found, serum was absorbed with Protein A Sepharose CL 4B (Pharmacia) and rerun in the EIA test. Relative EIA titers were calculated as follows: Sample absorbance - low standard absorbance high standard - low standard Culture X 100 Specimens were placed in 2 M sucrose-phosphate medium for transport and when possible cultured immediately on arrival to the laboratory. When this was not possible, the specimens were frozen at -70 C to await culture, which was never done >4 days after arrival at the laboratory. Culture was performed on cycloheximide treated McCoy cell monolayers, freshly prepared for each inoculation. Three hundred microliters of agitated specimen were added to each well of a 24-well plate and centrifuged at 1,800 X g for 1 hour at 35 C. Plates were then incubated in the presence of 5% CO2 for 48 hours before aspiration of the culture media and fixation with ethanol. Staining was done with Chlamydia Culture Confirmation monoclonal (Syva Co.) and plates were read using a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) at 100X and 400X magnification. Positive controls were run in parallel using clinical strains, not typed. Chlamydia quality control testing is employed at regular intervals with specimens sent out from the National Bacteriology Laboratory. All cultures were read after primary inoculation. No blind passages were done. Micro Trak MicroTrak tests were collected and fixed as described above. All MicroTrak slides were frozen at -20 C on arrival at the laboratory. They were brought to room temperature, dried and 50 ~L of MicroTrak monoclonal antibody suspension for the direct test was applied to each slide. The slides were incubated for 20 minutes in a moist chamber at room temperature, gently rinsed with distilled, deionized water, and air dried. The slides were read at 400X and 1,000X magnification. Interpretation was done according to the manufacturers instructions using positive reference slides in parallel to all readings. Elementary bodies were counted in every smear, unless they were too numerous to permit accurate counts (>50). All slides were read by one of two experienced microbiologists who were unaware of other test results. Slides with 10 or more EBs were regarded as positive as recommended by the manufacturer. Slides with lower numbers were regarded as suspected positive. Chlamydiazyme Specimens for Chlamydiazyme were collected as described above, and tested within 7 days of collection. Results were expressed as absorbance values at 492 nm. Pharmacia Chlamydia EIA Specimens were taken and tests performed according to the manufacturers instructions. Absorbances were read at 405 nm. 306 Thejls et al. Persistent chlamydia and infertility

4 Table 1 Serum IgG Antibodies in Relation to Findings at Operation and in Pregnant Women Group EIA~ 20" MIF ~ 64" Prior PID (n = 131) 68 (52)b 79 (60)e Adhesion (n = 35) 9 (26) 12 (34) Normal (n = 90) 11 (12)d 14 (16)" Pregnant (n = 1,078) 86 (8) 143 (13) "Values are no. with percents in parentheses. b The difference between the prior PID group and the normal group is significant (P < 0.001). e The difference between the prior PID group and the normal group is significant (P < 0.001). d The difference between normal group and pregnant group is not significant. " The difference between normal group and pregnant group is not significant. Statistical Methods The X 2 test and Fisher's exact test were used for comparisons between patient groups. The twotailed pooled variance t-test was used to compare values of means. Spearman's rank correlation coefficient was used for evaluation of correlation between different serological methods. History RESULTS One or more episodes of verified or suspected PID was reported by 78 patients. Forty-nine (63%) ofthese had signs of prior PID at operation. Ten of the 78 (12.8%) had had one or more tubal pregnancies. The geometric mean titer and the number of patients with positive chlamydia serum antibodies among the patients with a history of PID did not differ from those of 178 patients without a history of PID. Patients with silent PID (prior PID group without a history of PID) did not differ in mean titer or number of seropositive patients from the group with overt clinical PID (prior PID group with a history of PID), but in only a few patients with a history of PID had the diagnosis been verified laparoscopically. Chronic pain was reported by 15 of 78 patients (19%) with a history of PID and in 8 of 178 (4%) without PID in the history (P < 0.001). Signs of prior PID but without history of PID chronic pain was reported by 3 of 82 patients as compared with 10 of 90 with normal pelvic findings. A history of verified chlamydia infection (positive culture) was reported by 30 patients. Twenty of these (67%) had postinfiammatory damage Vol. 55, No.2, February 1991 to the tubes, but only 8 had a history of suspected PID. Findings At Operation Signs of prior PID were found in 131 patients. Normal tubes were found in 90 cases and adhesions from prior pelvic surgery, endometriosis, or of unknown etiology were found in 35 patients. In the prior PID group, serum IgG against chlamydia, as measured by MIF, was found in a titer of 1/64 or more in 79 (60%) of 131 patients compared with 14 (16%) of 90 patients with normallaparoscopic findings (P < 0.001). Relative EIA titer of20 or more was found in 68 (52%) of 131 patients in the prior PID group and in 11 (12%) of 90 patients with normal findings (P < 0.001). Pregnant women had antibodies against chlamydia in the same prevalence as infertile women with normal tubes (Table 1). These findings hold true even when the material is grouped in age cohorts (Table 2). Only 48 (36%) of 131 patients with prior PID had a history of suspected PID. Women with prior tubal ectopic pregnancies (EPs) had findings of prior PID in 20 (71 %) of 28 cases. Antibodies against chlamydia were found in 16 of these pa- Table 2 Number of Age-matched Patients in Prior PID Group, Normal Group, and Pregnant Women who Have Positive Serum Titers" Age, years Infertile infertile Infertile normal Pregnant in cohort priorpid b tubes b women b <25 (n = 12) 2/4 (50) 0/5 (0) 29/312 (9) 25 to 27 (n = 46) 16/27 (59)e 3/16 (19)d 25/242 (10) 28 to 30 (n = 53) 18/28 (64)" 1/17 (6)d 40/208 (19) 31 to 33 (n = 70) 21/32 (66)1 7/29 (24)d 30/162 (19) >33 (n = 75) 22/40 (55)B 3/23 (13)d 19/154 (12) Total 131 (100) 90 (100) 1078 (100) " Antichlamydial antibodies of ~64 inverse titer as measured bymif. b Values are no./total no. with percents in parentheses. e The difference between prior PID group and normal group is significant (P = using Fisher's exact test). d There was no significant difference between normal group and pregnant women. "Prior PID group and normal group differs significantly (P = ). 1 Prior PID group and normal group differs significantly (P = ). g Prior PID group and normal group differs significantly (P = ). Thejls et al. Persistent chlamydia and infertility 307

5 Table 3 Findings in 9 Patients Positive in Culture, Two or More Direct Tests, or With >26 EBs in MicroTrak Patient no. and group MicroTrak EBs Chlamydiazyme 1. PriorPID ;;;,:50T b neg 2. Adhesion ;;;,:50E neg ;;;,:50C 3. PriorPID ;;;,:50E neg ;;;,:50C 4. PriorPID 10E neg 5. Normal ;;;,:50C neg 6. Normal ;;;,:50C pos 5E 7. Normal ;;;,:50C pos 8. PriorPID 40C neg 4E 6T 9. PriorPID 26C neg The direct EIA test from Pharmacia. tients. Of 131 patients with prior PID, 72 (55%) were secondarily infertile compared with 33 (37%) of90 patients with normal tubes (P < 0.025). Ifpatients who had only EP are excluded, the figures are 63/122 (51%) and 32/89 (36%), respectively (P < 0.05). Cultures And Direct Tests Positive cultures were obtained from 4 patients, positive chlamydiazyme test in 2 patients, and positive Pharmacia direct EIA test in 2 patients. Ten or more EBs were found in 21 (8.2%) of 256 patients. The lowest count of EBs with positive culture was 26. The 4 patients with positive EIA antigen tests had all >26 EBs. The relation between EB counts, culture, and the two EIA tests for patients with >26 EBs is shown in Table 3. Patients with findings of >5 EBs at any site are listed in Table 4 with antibody titers and the location of the EBs. Any EB (~1 EBs) was found in 42 (16%) of256 patients. In the prior PID group, 14 (11 %) of 131 patients had 10 or more EBs as compared with 4 (4%) of90 patients with normal tubes (NS, P = 0.1). If patients with counts of 1 to 9 EBs are included, 26 (20%) in the PID group and 8 (9%) in the normal group had any EB (P = ). If we assume that a finding of 10 or more EBs in the MicroTrak test is safely regarded as a true positive test, the sensitivity of our culture method used in this population is only 19% (4/21) and the negative predictive value 93% (235/252). Serology Patients with positive MicroTrak test (~10 EBs) had negative or borderline IgG titers with the EIA EIA pharmacia Culture IgG-EIA IgG-MIF neg neg neg neg 0 64 pos neg neg posc neg posc 0 32 neg posc+e neg neg neg neg pos posc 0 64 b The location of the elementary bodies marked with T, fallopian tubes; E, endometrium; and C, cervix. and MIF test in 11 (52%) and 8 (38%) of 21 cases, respectively. Immunoglobulin M serum titers of ~20 relative EIA titer in the EIA test were found in 23 patients of whom only 1 had positive MicroTrak but negative culture and direct EIA test. Tubal specimens were missing from 18 of these patients. Seven patients had a serum IgM titer of ~16 in the MIF test. Six of these had even positive IgM titers in the EIA test but none had chlamydia in Table 4 Patients With EBs in Descending Order of Number of EBs and Their Antibody Titers MicroTrak no. of EBs SerumIgG Patient Classification no. C E T at operation EIA MIF ND b PriorPID ND PriorPID ND PriorPID ND Normal ND Adhesion PriorPID PriorPID ND PriorPID ND PriorPID ND PriorPID ND PriorPID ND Adhesion Adhesion PriorPID ND PriorPID ND Adhesion ND Normal ND Prior PID ND PriorPID Location ofebs marked with C, cervix; E, endometrium; T, fallopian tubes. b ND, not done. 308 Thejls et al. Persistent chlamydia and infertility

6 any of the antigen tests. Tubal specimens were missing in 4 of the 7 patients. The EIA method and the MIF method, type D-K, correlated in the following way: both tests were positive in 66% (165/252) of the specimens. Positive titer in one test and borderline in the other was found in 23% (58/252) and negative titer in one and positive in the other in 12% (29/252). The Spearman rank correlation (rho) was Analysis of Cross-Reaction Between C. pneumonia Serum Titers and EIA and MIF, type D-K Titers For serum specimens with EIA test > 0 (n = 127), the Spearman rank correlation between EIA titers and titers against C. pneumonia (MIF) was For specimens with MIF titers against type D-K ~ 32 (n = 156) the correlation was 0.09 when tested against C. pneumonia titers. No crossreaction with neither the EIA titers nor the MIF, type D-K titers could therefore be observed. DISCUSSION Our study confirms that the prevalence of serum antibodies against chlamydia is much higher in infertile women with signs of prior PID than in infertile women with normal pelvic findings and in pregnant women. We found that women with tubal factor infertility had a history of PID in only one third of the cases in accordance with many other studies.9-12,17 This gives further support for the view that silent infections are the most common cause of tubal factor infertility. No difference in prevalence of serum antibodies and mean titers was found between women with signs of prior PID with history ofpid (overt PID) and those without (silent PID). This confirms the findings of Patton et al.18 who also demonstrated the same electron microscopical changes after overt and subclinical salpingitis. Secondary infertility was more common in the prior PID group than in the group with normal tubes in accordance with the studies of Osser et al.9 and Moore et al.p giving some support for the view that vaginal delivery and abortion might be a risk factor for acquiring ascending genital infection. Our most remarkable finding was the high preva- 1ence of chlamydial elementary bodies in women with negative culture. The two obvious questions to be considered are whether the culture technique has lower sensitivity in this population than in others and to what extent misinterpretation ofthe MicroTrak test can explain the results. A single blind passage in tissue culture has been shown to improve the sensitivity in lower genital tract specimens, but multiple passages were not found to further improve the sensitivity.19 In the study of Shepard and Jones, 5 multiple passage was necessary to detect chlamydia in the tubes of infertile women and sensitivity was substantially improved in specimens from the endometrium. This was also the case in the study of Keilani et al. 6 Even in acute salpingitis it seems as if the sensitivity of culture for specimens from the endometrium and tubes is lower compared with cervical specimensy It is therefore possible that the use of only primary inoculation without blind passage may have contributed to the low isolation rate in our study. Interpretation of DF A slides with very few EBs is not without problems. Nonspecific staining of particles in the smear are common and some bacteria bind the fluorescent antibodies.20 These unspecific staining artifacts and bacteria are in general easily distinguished from chlamydial EBs by their shape, size, and intensity of color. Given the typical morphology and color of the EB and the relative ease of interpretation of slides with> 10 EBs, we believe that these slides represent true positive chlamydia tests and that the negative cultures are due to low numbers of EBs or host defense factors inhibiting the viability of the chlamydia. The outcome in this study may bean analogy to the findings in studies oftrachoma where active chlamydia infections without cultivable organisms have been demonstrated with the use of multiple nonculture methods.21 Although we should not uncritically accept tests with low counts of EBs, it is noteworthy that the finding of any EB (~1) is twice as common among women with prior PID than in infertile women with normal tubes. C. trachorriatis has been isolated from the tubes and endometrium from women with unexplained infertility and a possible causative role suggested but the documentation for this is still modest No conclusions can be drawn from the lack of correlation between the serum IgG findings and the MicroTrak test. A low grade infection with chlamydia does not necessarily elicit an immune response. 3 We conclude that persistence of C. trachomatis in infertile women is common but difficult to verify with routine tissue culture. The DF A test has in our study given higher positive rates than both culture and the two direct EIA tests. However, subjec- Vol. 55, No.2, February 1991 ThejIs et al. Persistent chlamydia and infertility 309

7 tive factors may interfere with the interpretation of slides containing very few EBs. The exact prevalence of C. trachomatis in infertile women can therefore not be settled solely by use of DF A tests. Use of polymerase chain reaction (PCR) have recently shown promising results in the diagnosis of genital tract infections with C. trachomatis. 25 In future studies, it may be useful to combine DF A tests with PCR to establish the true prevalence of persistent infections with C. trachoma tis in asymptomatic infertile women. Acknowledgments. We express our great gratitude to Goran Larsson, M.D., Katarina Hogset, M.D., and Bengt Andrae, M.D., at the Department of Obstetrics and Gynecology, Gavle Central Hospital, for their invaluable assistance. REFERENCES 1. Cates W: Sexually transmitted organisms and infertility: the proof of the pudding. Sex Transm Dis 11:113, Brunham RC, Maclean IW, Binns B, Peeling RW: Chlamydia trachomatis: its role in tubal infertility. 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