INFECTION WITH CHLAMYDIA TRACHOMATIS IN FEMALE COLLEGE STUDENTS

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1 AMERICAN JOURNAL OP EPIDEMIOLOGY VOL, Copyright 98 by The Johns Hopkins University School of Hygiene and Public Health Printed in U.S-A. All rights reserved INFECTION WITH CHLAMYDIA TRACHOMATIS IN FEMALE COLLEGE STUDENTS WILLIAM M. McCORMACK, - BERNARD ROSNER, DOROTHY E. McCOMB, JOHN R. EVRARD 3 AND STEPHEN H. ZINNER McCormack, W. M. (Box 6, Downstate Medical Center, Brooklyn, NY 3), B. Rosner, D. E. McComb, J. R. Evrard and S. H. Zinner. Infection with Chlamydla trachomatlt in female college students. Am J Epidemiol 98;:7-. Chlamydla trachomatia was isolated from genital specimens from (.9) of 3 female college students. Antibody to C. trachomatlt was found in the genital secretions of (.9) of 37 women. Multiple logistic regression analysis showed race, number of sexual partners, and use of barrier methods of contraception to be predictive of infection with C. trachomatlt. Logistic regression analysis found race, number of sexual partners, use of barrier methods of contraception, and presence of cervical erythema to be predictive of local chlamydial antibody. White participants were infected less often ( of 388 (3.)) than Mack participants (9 of 3 (.9)) (p <.) and were less likely to have local chlamydial antibody. None of the sexually inexperienced women were infected or had local antibody. Among the sexually experienced women, chlamydial infection and local chlamydial antibody increased with increasing number of sexual partners only for women who were not using barrier methods of contraception. Sexually experienced women who used barrier methods of contraception (condom, diaphragm) were less likely to be infected (one of (.)) than were sexually experienced women who used other contraceptive measures or who did not use contraception ( of 76 (7.)) (p =.3). Women who used barrier methods of contraoeption also were less likely to have local chlamydial antibody. Women with cervical erythema were more likely to have local chlamydial antibody ( of (36.)) than women without cervical erythema (8 of 6 (.3)). Vaginal colonization with other sexually transmitted microorganisms {Uycoplatma homlnla, Unaplaama urealytlcum, Trichomonas vaglnalfo) was noted more often among women with chlamydial infection than among uninfected women. chlamydia; chlamydial infections; contraception Chlamydia trachomatis has emerged as a major genital pathogen (). It is now well established as the cause of about per cent of cases of nongonococcal urethritis (). Recent studies have implicated this Received for publication January, 983, and in * Reprint requests to Dr. McCormack at present final form May 8, 98. address: Box 6, DownBtate Medical Center, Channing Laboratory, Brigham and Women's Clarkson Avenue, Brooklyn, NY 3. Hospital, and Department of Medicine, Harvard Med- Supported by a research grant (AI 38) from the ical School, Boston, MA. Department of Microbiology, Harvard School of National Institute of Allergy and Infectious Diseases and by a research grant (HD 9) from the National Public Health, Boston, MA. Institute of Child Health and Human Development. ' Division of Infectious Diseases, Department of The authors are indebted to Dr. Julius Schachter Medicine, Roger Williams General Hospital, and Di- and Dr. Roger L. Nichols who offered many helpful vision of Biological and Medical Sciences, Brown Uni- suggestions. The authors gratefully acknowledge the versity, Providence, RI. assistance of Vicki Ann Crockett and Susan Alpert. Downloaded from at Pennsylvania State University on March, 6 7

2 8 McCORMACK ET AL. organism as a cause of acute epididymitis in young men (3), pelvic inflammatory disease (), and neonatal pneumonia (). Despite increasing awareness of the importance of infections with C. trachomatis, little is known about the prevalence and natural history of this infection in women. During the academic year, we examined female college students for a number of sexually transmitted conditions. Previous publications have described infections in this population with Group B Streptococcus (6) and Gardnerella vaginaus (Corynebacteriwn vaginale) (7). In addition, there has been a preliminary report describing chlamydial infections in the first 8 women who were studied (8) and a publication describing the long-term follow-up of some of the women who were infected with C. trachomatis (9). The present report describes chlamydial infections in the entire population. MATERIALS AND METHODS Women presenting to the only gynecologist in the student health service of a large university were invited to participate in the study regardless of their reason for visiting the physician. About three-quarters of the participants came to see the gynecologist for fertility control or for routine physical examinations and cervical cytologic examinations (or for both). Participants were asked to complete a questionnaire containing demographic questions as well as questions on sexual development, sexual experience, and gynecologic symptoms. All participants were examined by the same physician. During the examination, a culture for C. trachomatis was obtained from the endocervical canal, ectocervix, and posterior fornix with a single cotton-tipped applicator, which was placed into. ml of sucrose-phosphate solution () containing streptomycin ( /*g/ml), nystatin ( ng/ ml), and gentamicin ( /ig/m\). These specimens were kept on wet ice for 3-6 hours, frozen, and stored at 8 C. A sample of endocervical secretions was obtained with a urethrogenital Calgiswab (Inolex Corporation, Glenwood, IL), the end of which was cut to fit into a /il plastic tube. These specimens were handled in the same manner as the cultures. To ensure anonymity of the participants, the questionnaire and specimens were identified only by a code number. All specimens were tested without reference to personal data or results of completed tests. After thawing at 37 C, each sample was homogenized with a syringe and needle and examined for C. trachomatis with use of a micro-cell culture method (). Duplicate. ml samples of the specimen were inoculated into wells containing irradiated baby hamster kidney cells (BHK-), obtained from Dr. William A. Blyth, then at the Lister Institute in London. These cells were selected because they were found to be more susceptible to C. trachomatis infection than McCoy cells (). A serotype B C. trachomatis strain of standard laboratory passage was used as a positive control. After centrifugation at, g for 3 minutes at 33 C, the supernatant was replaced with modified rubella medium () containing the same antibiotics used in the collection of samples. After incubation of these cultures at 3 C for hours, both cover glasses from each sample were rinsed with buffered.8 per cent NaCl, fixed in acetone, and mounted cell side up on a glass slide with Permount fixative (Fisher Scientific Co., Boston, MA). After staining with fluorescein-labeled human antiserum to lymphogranuloma venereum agent, which reacts with all recognized serotypes of C. trachomatis, the cells were examined for inclusions. Antibody tests on cervical secretions were done by the immunofluorescence method. The antigen was composed of a pool of serotypes of C. trachomatis. The serotypes and strains used were A/HAR- 3, B/HAR-36, C/HAR-3, D/MRC-, E/ CAL-, F/CAL-9, G/UW-7, H/UW-3,/ UW-, Lj/, W3, and La/. Equal portions of per cent infected yolk sac suspensions were pooled and smeared over Downloaded from at Pennsylvania State University on March, 6

3 CHLAMYDIAL INFECTIONS IN WOMEN 9 an entire microscope slide with use of the edge of a second slide. After fixation in acetone, the antigen surface was scored both by a diamond pencil and by a sharp wooden stick, which served both to identify the areas to be used and to prevent dissipation of the test samples during wet chamber incubation. These areas were about mm in diameter. Cervical secretions were eluted with fil of phosphate-buffered saline (an assumed dilution of :). The pooled C. trachomatis antigen was first exposed to cervical secretions and, after rinsing of the slides, exposed further to a :3 dilution of fluorescein-conjugated rabbit antibody to human globulin (Clinical Sciences Co., Whippany, NJ). The conjugate was standardized in a block titration with high-titered (:,6) human antiserum to lymphogranuloma venereum agent; this standard contained -8 units of antibody. Titration of cervical secretions was not possible because of the limited amount of sample available. Previously described methods were used to examine vaginal specimens obtained with use of sterile cotton-tipped swabs for Mycoplasma kominis (3), Ureaplasma urealyticum (3), Group B Streptococcus (6), and G. vaginalis (formerly known as C. vaginale and Haemophilus vaginalis) (7). For the identification of Trichomonas vaginalis, vaginal swab specimens were inoculated into Trichosel broth (Baltimore Biological Laboratories, Cockeysville, MD) which was incubated at 37 C. Aliquots were examined microscopically for motile trichomonads daily for seven days. If yeast-like organisms were noted in the Trichosel broth, an aliquot was inoculated onto blood agar and examined with use of routine methods. A vaginal swab specimen was inoculated onto blood agar, incubated at 37 C for 8 hours in a candle jar and examined for bacteria and fungi with use of standard methods. A participant was considered to be colonized with Candida species if these organisms were isolated from the Trichosel broth or on the primary blood agar plate (or both). The Yates' corrected chi-square test and Fisher's exact test were used for analysis of x contingency tables in table 6 (). Mean number of years of parental education were compared for white women and black women and for infected and uninfected women with use of the two-sample t-test with equal variances (). StepwiBe logistic regression methods () were used to control for the effects of several variables in predicting chlamydial infection and local chlamydial antibody. Variables were added to the regression model until no further variables entered the model at a significance level of p <.. RESULTS Genital specimens from 8 of the young women who participated in the study were available for examination for C. trachomatis. Forty-five (9.8 per cent) of the 8 women had been pregnant, but only two women were knowingly pregnant when they were studied. Seven of the women were of races other than white or black, were of unknown race and an additional nine women were missing values for at least one of the key independent variables considered in the multiple logistic regression analysis listed below. We chose the 3 women of white or black race with complete data on all key independent variables as the population for study of infection with C. trachomatis. C. trachomatis was isolated from (.9 per cent) of the 3 participants. The mean age of the infected participants (. ±. years; mean ± standard error) did not differ significantly from the mean age of those who were not infected (.6 ±. years). Most of the women with chlamydial infections were asymptomatic. Two ( per cent) of the complained of dysuria. Three ( per cent) reported abdominal pain and seven (33 per cent) had noted an abnormal vaginal discharge. Noninfected women re- Downloaded from at Pennsylvania State University on March, 6

4 McCORMACK ET AL. ported these symptoms with similar frequency. A stepwise multiple logistic regression analysis was performed with chlamydial infection as the dependent variable (). The following independent variables were included: age, race, class (freshman, sophomore, etc.), recent use of antibiotics, day of menstrual cycle, age at menarche, use of tampons or sanitary napkins, history of pregnancy, symptoms of dysuria, lower abdominal pain, or vaginal discharge, use of oral contraceptives, intrauterine devices, condoms or diaphragms, lifetime number of male sexual partners, number of male sexual partners during the past year, frequency of intercourse during the past week, interval between last intercourse and examination, cigarette smoking, marital status, mother's formal education, father's formal education, practice of fellatio, cunnilingus, anal intercourse, or douching, abnormal vaginal discharge, or cervical erythema noted during the physical examination. Race, number of male sexual partners, frequency of intercourse during the past week, interval between last intercourse and examination, and method of contraception were considered to be key variables. Participants with missing information on any of the key variables were excluded from the multiple logistic regression analyses. For all other variables, missing values were assigned to the most frequent category (for discrete variables) or to the median value (for continuous variables). In addition, interactive effects were considered between variables relating to sexual experience and contraceptive method. Table shows the variables that were significantly predictive of chlamydial infection. These infections were less likely to occur among whites (p <.). In addition, women who did not use barrier methods of contraception were more likely to have chlamydial infection with an increasing number of sexual partners (p =.). No such relationship was detected for TABLE l Independent variables predictive of chlamydial infection among female college students in a multiple logistic regression analysis, n = 3 Independent variables* Constant Race White Black Lifetime number of sexual partners for users of nonbarriert methods of contraception! 3 Odds ratio Regression coefficient Standard error p value.9 <... * Listed in order of entering the logistic regression model. t Barrier methods of contraception include diaphragm, diaphragm and foam, and condom; nonbarrier methods include all other methods of contraception or no contraception. X This variable is defined as for users of barrier methods of contraception. women who used barrier methods of contraception. Results are presented on local antibody to C. trachomatis for 37 women who were of white or black race and had no missing key values for any of the independent variables listed above. Antibody to C. trachomatis was detected in the genital secretions of (.9 per cent) of 37 participants. Local chlamydial antibody was detected in the secretions of 7 (86. per cent) of women from whom C. trachomatis was isolated and from 33 (8.3 per cent) of 396 women who had negative chlamydial cultures (p <.). Chlamydial cultures were not done for women, two of whom had local antibody. A logistic regression analysis was also performed with local chlamydial antibody as the dependent variable and with the independent variables listed above. This analysis is summarized in table. Predic- Downloaded from at Pennsylvania State University on March, 6

5 CHLAMYDIAL INFECTIONS IN WOMEN TABLE Independent variables predictive of local chlamydial antibody among female college students in a multiple logistic regression analysis, n = 37 Independent Odds Rc F e8 ~ sion Stan.,. variables* ratio coeffidard cient error pvalue Constant -.9 Race White <. Black. Lifetime number of sexual partners for users of nonbarrierf methods of contraception^..6.9 < Cervical erythema Yes No. * Listed in order of entering the logistic regression model. t Barrier methods of contraception include diaphragm, diaphragm and foam, and condom; nonbarrier methods include all other methods of contraception or no contraception. t This variable is defined as for users of barrier methods of contraception. TABLE 3 Relation of race to infection with Chlamydia trachomatis and to local chlamydial antibody among female college students Isolation of C. trachomatis Local chlamydial antibody Race C. trachoma- Chlamydial ri, isolated «**«& studied studied Present White Black Total tive variables included race (p <.), lifetime number of sexual partners among women who used nonbarrier methods of contraception (p <.), and presence of cervical erythema (p =.7). Data on chlamydial isolation and on local chlamydial antibody in each of the racial groups are presented in table 3. White participants were less likely to be infected with C. trachomatis than black participants (p <.). This organism was recovered from (3. per cent) of 388 white women and from nine (.9 per cent) of 3 black women. White participants were also less Likely to have local chlamydial antibody (p <.). To assess socioeconomic status, we tabulated parental education. The fathers and mothers of the black participants had significantly less formal education (mean ± standard error of.6 ±.7 and 3. ±.6 years) than the fathers and mothers of the white participants (7. ±. and. ±. years) (p <.). Among the black women, however, no significant association was found between parental education and infection with C. trachomatis. The fathers and mothers of the infected black women had.8 ±.9 and. ±.8 years of formal education; the fathers and mothers of the uninfected black women had.9 ±.7 and.9 ±.7 years of formal education. In tables and, we show the relationship between sexual experience and infection with C. trachomatis and presence of local chlamydial antibody, respectively, after controlling for race and method of contraception. None of the sexually inexperienced women were infected and none of the 8 sexually inexperienced women with data available had local chlamydial antibody. Among those who had experienced sexual intercourse and did not use barrier methods of contraception, the prevalence of infection with C. trachomatis and of local chlamydial antibody rose in relation to the number of lifetime sexual partners. In contrast, this trend was not apparent for either outcome variable for either race in women who used barrier methods of contraception. Furthermore, strong racial differences exist with respect to both infection with C. trachomatis and local chlamydial antibody after controlling for number of sexual partners and method of Downloaded from at Pennsylvania State University on March, 6

6 McCORMACK ET AL. TABLE Relation of sexual experience to infection with Chlamydia trachomatis among female college students by race and method of contraception Lifetime no. of sexual DArtncrfl pal biici o Black women' 3-6 or more White woment 3-6 or more Oral contraceptives positive Barrier methods positive * Other methods of contraception (including no contraception) positive.... * There were four black women who had had no sexual partners; none of these women were infected with C. trachomatis. t There were 6 white women who had had no sexual partners; none of these women were infected with C. trachomatis. TABLE Relation of sexual experience to local chlamydial antibody among female college students by race and method of contraception Lifetime no. of sexual Black women* 3-6 or more White woment 3-6 or more Oral contraceptivt positive Barrier methods positive Other methods of contraception (including no contraception) 7 7 positive.... * There were three black women who had had no sexual partners; none of these women had local chlamydial antibody. t There were white women who had had no sexual partners; none of these women had local chlamydial antibody. contraception. Finally, there was a positive association between the presence of cervical erythema and local chlamydial antibody after controlling for race, number of sexual partners, and method of contraception. Four of (36. per cent) women with cervical erythema had local chlamydial antibody as opposed to 8 of 6 (.3 per cent) women without cervical erythema (p =.63). Table 6 compares rates of colonization with other genital microorganisms among the women who were infected with C. trachomatis and the uninfected women. Sexually transmitted organisms such as M. hominis, U. urealyticum, and T. vaginalis were found statistically significantly more often among the women with chlamydial infections. Normal vaginal organisms {Candida species, Escherichia coli, Staphylococcus epidermidis) were found about as often in the infected as in the uninfected women. Women who were infected with C. trachomatis were more likely than unin-

7 CHLAMYDIAL INFECTIONS IN WOMEN 3 TABLE 6 Colonization with other genital microorganisms among women infected with Chlamydia trachomatis and among uninfected women Microorganism Infected with C. trachomatis : isolated Not infected with C. trachomatis isolated (two-tailed) Mycoplasma hominis Ureaplasma urealyticum Trichomonas vaginalis Gardnerella vaginalis Group B Streptococcus Candida species Escherichia coli Staphylococcus epidermidis * * Per cent of women colonized with indicated microorganism. t NS, not statistically significant. fected women to be colonized with G. vaginalis (p =.3). DISCUSSION The importance of genital infection with C. trachomatis has been stressed by a series of recent publications (-). However, chlamydial diagnostic services are not generally available. Thus, many clinicians, unable to make a definitive diagnosis of chlamydial infection, tend to regard these organisms as rare or unusual. The data presented herein show that this is not at all the case. Infections with C. trachomatis were documented in.9 per cent of 3 unselected female college students presenting, mostly for routine examinations, to the gynecologist in their student health service. Other studies () have shown that genital chlamydial infection is even more prevalent among women presenting to sexually transmitted disease clinics and among women receiving prenatal care at public hospitals in urban areas. The method () we used (micro-cell culture with BHK- cells) may have been less sensitive than the use of cycloheximide-treated McCoy cells () which has emerged as the method of choice for chlamydial isolation. Thus, we may have underestimated the prevalence of chlamydial infection. This does not, however, detract from the validity of the internal comparisons * <. <..9.3 NSt NS NS NS With use of multiple logistic regression analysis, we were able to show that white race is associated with a decreased likelihood of infection with C. trachomatis, while having many lifetime sexual partners is associated with an increased likelihood of infection with C. trachomatis in women using nonbarrier methods of contraception. A similar analysis showed that white race, the lifetime number of sexual partners in women using nonbarrier methods of contraception, and the presence of cervical erythema are associated with the presence of local chlamydial antibody. These variables are statistically related to chlamydial infection and to the presence of local chlamydial antibody, respectively, in individual cross-tabulations. The multiple logistic regression analysis indicates that each of these variables is independently predictive of chlamydial infection and of local chlamydial antibody. These data presented herein indicate that black women are considerably more likely than white women to be infected with C. trachomatis and to have local chlamydial antibody. Blacks are known to be at greater risk for other sexually transmitted infections (). The observation that nongonococcal urethritis is relatively more common than gonococcal urethritis among white men (6) has led to the erroneous impression that nongonococcal urethritis in par- Downloaded from at Pennsylvania State University on March, 6

8 McCORMACK ET AL. ticular and chlamydial infection in general are more common among whites than among blacks. As indicated by the present data, this is certainly not the case among college women. In this group, blacks bear a disproportionate portion of the burden of chlamydial infections. C. trachomatis, like Neisseria gonorrhoeae and T. vaginalis, is a classic exogenous sexually transmitted organism. C. trachomatis was not isolated from any of the sexually inexperienced women in this population. Nor was local chlamydial antibody, which presumably reflects current or past chlamydial infection, present in any of the sexually inexperienced women. This suggests that local chlamydial antibody is a more specific indicator of prior genital chlamydial infection than serum antibody which was present in about per cent of the sexually inexperienced women in this population (8). Among the women who were sexually experienced the presence of C. trachomatis and of local chlamydial antibody increased in relation to the number of lifetime sexual partners in women using nonbarrier methods of contraception. Since C. trachomatis primarily infects the cervix in women (), it is not surprising that women using barrier methods of contraception condoms and diaphragms did not show a pattern of increased risk with an increasing number of sexual partners. Other investigators (7-9) have found that women who used oral contraceptives were more likely than women who used other methods of contraception to be infected with C. trachomatis. The lower chlamydial infection rates among women who did not use oral contraceptives in those studies may have been due to use of barrier methods of contraception by some of the women. However, these data were not provided. In the present study, oral contraceptive users do not appear to be at increased risk of chlamydial infection compared with other users of nonbarrier methods. The sample size, however, is too small to allow meaningful comparisons. REFERENCES. Schachter J. Chlamydial infections. (Parts,, and 3.) N Engl J Med 978;98:8-3; 9-; Holmes KK, Handsfield HH, Wang SP, et al. Etiology of nongonococcal urethritis. N Engl J Med 97;9: Berger RE, Alexander ER, Monda GD, et al. Chlamydia trachomatis as a cause of acute "idiopathic" epididymitia. N Engl J Med 978;98:3-.. Mardh P-A, Ripa T, Svensson L, et al. Chlamydia trachomatis infection in patients with acute salpingitis. N Engl J Med 977;9 : Beem MO, Saxon EM. Respiratory-tract colonization and a distinctive pneumonia syndrome in infants infected with Chlamydia trachomatis. N Engl J Med 977;96: Baker CJ, Goroff DK, Alpert S, et al. Vaginal colonization with Group B Streptococcus: A study in college women. J Infect Dis 977;3: McCormack WM, Hayes CH, Rosner B, et al. Vaginal colonization with Corynebacterium vaginale (Haemophilus vaginalis). J Infect Dis 977;36: McComb DE, Nichols RL, Semine DZ, et al. Chlamydia trachomatis in women: antibody in cervical secretions as a possible indicator of genital infection. J Infect Dis 979;39: McCormack WM, Alpert S, McComb DE, et al. Fifteen-month follow-up study of women infected with Chlamydia trachomatis. N Engl J Med 979;3:3-.. Bovarnick MR, Miller JC, Snyder JC. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J Bacteriol 9:9:9-.. McComb DE, Puzniak CI. Micro cell culture method for isolation of Chlamydia trachomatis. Appl Microbiol 97;8: Rota TR. Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK- cells. In Vitro 977;3: Braun P, Klein JO, Lee YH, et al. Methodologic investigations and prevalence of genital mycoplasmas in pregnancy. J Infect Dis 97;:39-.. Armitage P. Statistical methods in medical research. New York: John Wiley, 98.. US Department of Health, Education, and Welfare. VD fact sheet 97: basic statistics of the venereal disease problem in the United States. DHEW publication (HSM) Atlanta: Center for Disease Control, Volk J, Krau8 SJ. Nongonococcal urethritis. A

9 Downloaded from at Pennsylvania State University on March, 6 CHLAMYDIAL INFECTIONS IN WOMEN venereal disease as prevalent as epidemic gonor- ogical and clinical correlates of chlamydial infecrhea. Arch Intern Med 97;3:-. tion of the Mrvili Br j Vener Dis 9 8 i ; 7: Svensson L, Westrom L, Mardh P-A. Chlamydia, a v., nr3...,... n....,,,.. ',. ii 9- Kmehorn GR, Waugh MA. Oral contraceptive v use trachomatis in women attending a gynecological,, ^ ^ ~. _,,. outpatient clinic with lower genital tract infection. ^ Prevalence of infection with Chlamydia tra- Br J Vener Dis 98;7:9-6. chomatis in women. Br J Vener Dis 98;7:87-8. Arya OP, Mallinson H, Goddard AD. Epidemiol- 9.