THE JOURNAL OF INFECTIOUS DISEASES • VOL. 131, NO.4' © 1975 by the University of Chicago. All rights reserved.
APRIL 1975
Infection with Chlamydia Group A in Men with Urethritis Due to Neisseria gonorrhoeae J. D. Oriel, P. Reeve, B. J. Thomas, and C. S. Nicol
From the Department of Venereology, St. Thomas' Hospital, and the Department of Bacteriology, University College Hospital Medical School, London, England
After treatment for urethritis caused by N eisseria gonorrhoeae, a proportion of male patients develop postgonococcal urethritis (PGU); their urethral discharge recurs and an excess of polymorphonuclear leukocytes is seen on microscopy of urethral material, but cultures for N. gonorrhoeae are negative. The possible causes of PGU have been discussed by Holmes et al. [1]. Although a few cases may be due to slow resolution of urethral inflammation or possibly to the emergence of gonococcal L-forms, it is thought that in the majority of men PGU is due to an associated nongonococcal urethritis (NGU), the two infections, gonorrhea and NGU, having been contracted at the same time. The longer incubation period of NGU [2] explains why PG U appears when the gonococcal infection is subsiding. The clinical features of
PGU and NGU are similar, and both respond to treatment with tetracyclines; furthermore, if gonorrhea is treated with a tetracycline, the incidence of PG U is significantly lower [3]. Thus it seems likely that the agent or agents which cause NGU may cause a proportion of PGU also. The etiology of N GU is not fully understood at pre sent. The urethritis is again evidenced by an excess of polymorphonuclear leukocytes seen on microscopy of urethral material and by the absence of N. gonorrhoeae. A few cases may be caused by bacterial infection secondary to urethral trauma or disease, or by trichomonal, candidal, or herpetic infections. But the majority of cases of N GU cannot be attributed to these causes and are thought to be due to the sexual transmission of other pathogenic organisms. There is now evidence that Chlamydia group A (Chlamydia trachomatis) is an important cause of N G U. By the use of a sensitive cell-culture technique, Chlamydia has been recovered from approximately 40% of men with N GU, and from many of their female sexual contacts [4, 51. On the other hand, attempted isolations of Chlamydia from control groups of men without urethritis have given positive results in less than 5% of cases [5, 6]. Chlamydia may also be related to PGU. In a
Received for publication June 24, 1974, and in revised form December 4, 1974. We are grateful to Mr. J. M. McCaUig for technical assistance with serology. Gentamicin was kindly supplied by Roussel Laboratories, Ltd., Wembly Park, Middlesex, England. This work was supported by grants from the Medical Research Council (Great Britain). Please address requests for reprints to Dr. J. D. Oriel, Department of Venereology, S1. Thomas' Hospital, London, S.E. 1, England.
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Men with urethritis due to Neisseria gonorrhoeae were treated with gentamicin, which is inactive against Chlamydia. Urethral specimens were collected before treatment and one and two weeks after treatment and cultured for Chlamydia in irradiated McCoy cells. The overall incidence of chlamydial infection was 15 of 44 (34%). All of 15 Chlamydia-positive men and 11 of 29 Chlamydia-negative men (38%) developed postgonococcal urethritis two weeks after treatment. Pretreatment sera were examined by a complement fixation test and a simplified indirect fluorescent antibody (FA) test with a yolk sac-cultured antigen, strain SA2(t). Sera from all of 15 Chlamydia-positive men had titers of ~1:16 in the indirect FA test; 13 of 29 Chlamydia-negative men (45%) had positive tests. The complement fixation test was insensitive, detecting chlamydial antibodies in only one of 15 sera from Chlamydia-positive men. These results suggest that Chlamydia may cause many cases of postgonococcal urethritis.
Chlamydia in Gonococcal Urethritis
Materials and Methods
All the patients were seen in the Department of Venereology, St. Thomas' Hospital, London, between February 1 and April 30, 1973. On their first attendance at the clinic, after general and local examinations, men provided specimens as follows. A specimen of urethral secretion was taken with a wire loop and spread on a slide for immediate gram-staining and microscopy. A second urethral specimen was inoculated onto a culture plate for detection of N. gonorrhoeae. A third specimen was mixed on a slide with a drop of saline and immediately examined for Trichomonas vaginalis . The remaining urethral material was collected with a
cotton-wool meatal swab that was broken into a 2-ml screw-capped bottle of cell culture medium for culture of Chlamydia. Urine was passed into two glasses. Blood was collected for serology for syphilis and Chlamydia. When the diagnosis of gonococcal urethritis had been made, the patient was immediately treated with a single im injection of gentamicin (240 mg), Consent to the use of this drug was obtained after the patient had been warned that there was a slight risk of toxic reaction, although in fact none was experienced. The patient was advised to abstain from sexual intercourse and the consumption of alcohol for at least two weeks. Follow-up examinations were performed after two to three days, seven days, and 14 days. Specimens were normally collected at least 2 hr after micturition and were examined as on the patient's first attendance, but cultures for Chlamydia were repeated only on the seventh and 14th days after treatment. Assessment of PGU. The diagnosis of POD was made if there was evidence of significant urethritis more than seven days after treatment. Although many men with PGU had symptoms such as dysuria or urethral discharge, we did not rely on clinical symptoms or signs in making the diagnosis. Significant urethritis was defined in terms of the numbers of polymorphonuclear leukocytes per mean high-power field (x 100 objective) on a gram-stained urethral smear; occasionally a centrifuged specimen of first-catch urine was used similarly. A patient who showed more than 20 polymorphonuclear leukocytes per mean high-power field was considered to have significant urethritis. Laboratory methods. Culture for N. gonorrhoeae was performed on a selective medium [11J. Cell culture for Chlamydia group A was undertaken by centrifugation of clinical specimens onto irradiated McCoy cells, followed by incubation and examination for chlamydial inclusions; this method has been described [5]. Two serological tests were used to detect chlamydial antibodies: C F test with a microtiter technique and a Chlamydia group-reactive yolk-sac antigen, and a simplified indirect FA test, modified from the method of Wang and Grayston [12]. A yolk sac-cultured antigen, strain SA2 (0, was used. This was partially
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recent investigation Chlamydia was recovered from 29% of a group of men with gonococcal urethritis, and it was reported that after treatment with penicillin the incidence of PG U was higher in Chlamydia-positive than in Chlamydia-negative men [6]. However, penicillin inhibits the growth of Chlamydia [7], a fact that limits the value of studies of the relationships between Chlamydia and PG U in men treated with this drug. On the other hand, gentamicin, which is an effective treatment for gonorrhea [8], is known to have no in vitro action against Chlamydia [9]. In the present investigation, the relationship between Chlamydia and PGU has been studied in a group of men with gonococcal urethritis who were treated with gentamicin. Urethral specimens for cell culture for Chlamydia were collected before treatment and again one and two weeks after treatment, and the incidence of POD was compared in Chlamydia-positive and Chlamydia-negative patients. It is known that there is a serological response to genital chlamydial infection; antibodies have been detected by CF tests, FA techniques, and a radioisotope precipitation test [10]. During the present investigation, the first two of these methods were used to detect antibodies to Chlamydia in sera collected from the men with gonococcal urethritis on their first attendance in a clinic. Results of the serological tests were then compared with the results of isolation of Chlamydia.
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Oriel et al.
378
Results
Initially, 82 heterosexual men with confirmed gonococcal urethritis were investigated, but 38 were removed from the project for the following Table 1.
reasons: default, 16; initial contaminated specimen, eight; relapse of gonococcal urethritis after treatment with gentamicin, necessitating retreatment with another antibiotic, 9; reinfection with N. gonorrhoeae during follow-up, two; sera not available, three. This left 44 patients available for further study. Effect of repeated specimen collection on isolations of Chlamydia. Of the 44 patients studied completely, 11 (25%) yielded Chlamydia from urethral specimens collected on their first attendance. Of these 11 men, nine remained Chlamydia-positive after one and two weeks; one man was still positive after one week but became negative after two weeks; and one man, having still been positive after one week, gave a contaminated specimen after two weeks. However, four men who were initially Chlamydianegative yielded isolates subsequently, three of them after one week and one after two weeks (table 1). If the patients who yielded isolates at any time during the two-week study period are included, the incidence of urethral Chlamydia is raised to 15 of 44 men with gonococcal urethritis (34%).
Men with gonococcal urethritis who yielded isolates of Chlamydia on cell culture. First attendance
Patient no. I 2
History
Duration of symptoms (days)
G
]
G
A
One week after treatment
Chlam.
VD
PGV
Chlam.
+ +
+
+ + +
+ + + + + + + + + + +
3
2
4 5 6 7 8*
2 4 2 7
+ +
A
+ + + +
NGV
2]
9t lOt
21
]I
28
12
2
13
2 7
14 15
2
Two weeks after treatment
+ + +
+ + + + + + + +
+ + + + + + + +
+ +
+ + +
vn
PGV
Chlam.
+ +
+ + +
+ +
+
+ + +
+
+ + + + + + + + + + +
+ + + + + +
NOTE. A = asymptomatic; C = contaminated specimen; Chlam. = culture for Chlamydia; 0 nongonococcal urethritis; PO V = postgonococcal urethritis; V D = urethral discharge. * Patient also had trichomoniasis; he was treated with metronidazole. t· Patient had intercourse during follow-up.
=
C
+ C
+ + + + +
gonorrhea; NOV =
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purified by density gradient centrifugation and applied to Teflon-coated slides, dried, and fixed in acetone. This antigen was allowed to interact with serial twofold dilutions of test sera for 30 min at 37 C in a moist chamber, and then washed for 10 min with two changes of phosphate-buffered saline to remove uncombined serum. Fluorescein-conjugated antihuman globulin (Wellcome fluorescent antibodies, anti-human immunoglobulin-sheep, Wellcome Laboratories, London) was then applied to the preparation and allowed to react for 30 min at 37 C in a moist chamber; this procedure permitted detection of antigen-bound globulins. Fluorescence was observed with a Gillett and Sibert blue light fluorescence microscope. Phosphate-buffered saline was used as a diluent throughout. The serum titer was recorded as the highest dilution at which particulate fluorescence could be detected.
379
Chlamydia in Gonococcal Urethritis
Table 2.
Men with gonococcal urethritis who did not yield isolates of Chlamydia on cell culture. First attendance
Patient no.
16 17 18 19 20 21 22* 23 24 25t 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42t 43 44
History
Duration of symptoms (days)
G G,NGU G,NGU G,NGU NGU
3 2 1 3 1 5
G,NGU G G,NGU G,NGU G,NGU G,NGU NGU
A
NGU
Chlam.
UD
PGU
Chlam.
+ + +
C
+
UD
PGU
+
+ +
+
+
+ +
+
+
+
A
+ +
+ +
+
+
+ +
C
+
A
2
Chlam.
C
4 1 7 1 3 2 1 4
Two weeks after treatment
One week after treatment
C
1
1 5 3 14 3 6 3
+
+ C
+ +
+ +
C
C
2
NGU G,NGU
A I
+
+
NOTE. A = asymptomatic; C = contaminated specimen; Chlam. = culture for Chlamydia; G nongonococcal urethritis; PG U = postgonococcal urethritis; UD = urethral discharge. * Patient did not have PG U three weeks after treatment. t Patient had intercourse during follow-up.
=
gonorrhea; NGU
=
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Chlamydia isolations and PGU. Of the 15 men who gave chlamydia isolates on the first, second, or third attempt, significant urethritis (indicating PG U) was present one week after treatment in 13 patients and two weeks after treatment in all 15. After two weeks, nine of the 15 men with PGU had a manifest urethral discharge; the remaining six men had no discharge. Of the 29 men who were consistently Chlamydia-negative, significant urethritis (indicating PG U) was present one week after treatment in 12 and two weeks after treatment in 11 (38%). Of these 11 men, four had a manifest
Isolation of Chlamydia and duration of symptoms. The duration of symptoms (urethral discharge and dysuria) in the 15 men with gonococcal urethritis who yielded chlamydia isolates was one to 28 days (mean, 7.76 days; SD, 8.99); in the 29 men with gonococcal urethritis who were consistently Chlamydianegative, however, the duration of symptoms was one to 14 days (mean, 3.16 days; SD, 2.69) (table 2). This suggests that in men with gonococcal urethritis the duration of symptoms may affect the likelihood of isolation of Chlamydia.
Oriel et al.
380
Discussion
Chlamydia can often be recovered from the urethra of men with gonococcal urethritis. In a previous investigation we have found that of 104 valid urethral specimens from these men, collected on their first attendance, 22 (21 %) yielded Chlamydia in cell culture (authors' unpublished observations). This figure compares with the 25% rate of isolation found in the present study and with the results reported by other workers [6].
Not all the Chlamydia-positive men yielded isolates from the first urethral specimen. Little is known of the effect of repeated attempts at isolation on the isolation rate in either gonococTable 3.
cal urethritis or N GU, because patients with these diseases are normally treated on their first attendance with an antibiotic that may inhibit Chlamydia. There are two possible explanations for our results. Either our cell-culture system was not uniformly sensitive, or the chances of isolating Chlamydia from the urethra were increased by the passage of time, as the number of infective particles increases. The cell culture system did not appear to be insensitive, since we found that once patients had become Chlamydia-positive they usually remained so in subsequent specimens. We believe that the second supposition is more likely to be correct. This belief is strengthened by the observation that the mean duration of symptoms in the Chlamydia-positive group was double that in the Chlamydianegative group. A relationship between the isolation of Chlamydia from the urethra and the duration of symptoms in both gonococcal urethritis and N G U has been shown [6]. In investigating a possible connection between Chlamydia and PGU it is important that the gonococcal infection is treated with an antibiotic that has no action against Chlamydia. The choice is very limited, and gentamicin appears to be the most suitable drug. Not only is it reasonably effective for the treatment of gonococcal urethritis, curing about 90% of patients [13], but it has been shown to have no effect on Chlamydia in vitro; indeed, Wentworth has recommended its introduction into transport and growth media for Chlamydia to reduce bacterial contamination [9]. We have found that in our patients with
Antibodies to Chlamydia and incidence of postgonococcal urethritis (PG U).
Developing PGU (no. sera tested)
Indirect fluorescent antibody test positive*
Isolation of Chlamydia: result (no.)
No. (%)
GMT
History of GU or NGU
Positive (15)
15(100)
27
3
Negative (11) Positive (0)
2(18)
32
2
Negative (18)
11(61)
23
4
Yes (26)
No (18) NOTE.
GMT = geometric mean titer; GU = gonococcal urethritis; NGU = nongonococcal urethritis.
* Serum titers of
~1:16.
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urethral discharge. All patients with PGU were treated with a course of tetracycline, but were not studied further in the present investigation. Serology. PGU was diagnosed in a total of 26 men, of whom 15 had yielded chlamydial isolates on cell culture. All of these 15 gave positive results with the indirect FA test at titers of ~1:16, the geometric mean titer (GMT) was 1:27, and three of the men gave a past history of urethritis. None of the 18 men who did not develop PGU yielded chlamydia isolates; eleven of the 18, however, were seropusitive at titers of ~ 1:16, and four of these gave a past history of urethritis (table 3). In contrast with the indirect FA test, the CF test was insensitive. Only one of 15 sera from patients who had given chlamydia isolates on cell culture gave a positive result with the CF test.
Chlamydia in Gonococcal Urethritis
The antigen SA2(f), which is indistinguishable from the L GV II strain by immunotyping, has been shown by FA tests to give a one-way cross-reaction with antisera to Chlamydia group A, including serotypes D and E. This reaction, together with its ease of culture, led us to select S A2(f) as antigen for the indirect FA test. We are aware of the possible limitations imposed by this choice. As Wang and Grayston pointed out [15], our antigen is of "senior" type, capable of detecting antibodies to the B, D-E, and LGV immunotype complex; it may not detect antibodies to another commonly found genital serotype, the F-G complex, and probably would not react with antibodies to the more distantly related C, H, I, and K immunotypes. However, in our previous study [10], in which serotypes F and I were included as antigens, no sera were found that reacted solely with these antigens without also crossreacting with the LGV II immunotype complex. We have no information about the reactions of antibodies to the serotypes G, H, and the most recently discovered K. In practice, SA2(f) is apparently an efficient single antigen for measuring chlamydial antibodies in sera from men with NGU and PGU, although we would accept the possibility that this simplified system may fail to detect antibodies to some of the rarer serotypes. The serological results of the present investigation agree with those that we obtained from study of men with Chlamydia-positive NO U, 86% of whom gave positive results with the microimmunofluorescence test [10]. In both gonococcal urethritis and N G U, the great majority of patients who yield Chlamydia on cell culture give evidence of the presence of chlamydial antibodies in the serum. However, in both diseases a proportion of men who did not yield chlamydial isolates had antibodies to the predominant chlamydia serotypes. Of 29 sera from patients with gonococcal urethritis from whom Chlamydia was not isolated, 14 (48%) gave positive results with the indirect FA test. Some of these men gave a history of gonococcal urethritis or N G U and could thus have been exposed to genital chlamydial infection in the past. Others gave no such history, and we can only speculate on the significance of the positive serological results in these patients.
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gonococcal urethritis who have been treated with gentamicin, PGU regularly developed in those who were Chlamydia-positive before treatment. All of the 11 men who were Chlamydia-positive before treatment later developed POU. Similarly, four men who were initially Chlamydia-negative but who later gave isolates also developed POU. On the other hand, only 11 of 29 men (38%) who were consistently Chlamydia-negative developed POD after treatment. A relationship appears to exist between urethral Chlamydia and PGU. Two questions may now be asked. First, was Chlamydia sexually transmitted at the same time as N. gonorrhoeae, or was Chlamydia already present in the urethra and in some way activated by the gonococcal infection? Second, does Chlamydia cause PG U in these patients or is it merely associated with it, the true cause lying elsewhere? The first question cannot be answered completely until more is known of the epidemiology of chlamydial infection associated with gonorrhoea. We are studying this in a group of men with gonococcal urethritis and their sexual contacts. Although it has been argued that infection with N. gonorrhoeae may activate a latent chlamydial infection in the male urethra [6], our own studies have indicated that Chlamydia is not demonstrable in the urethra in the absence of urethritis [5], and we think it is more likely that N. gonorrhoeae and Chlamydia are sexually transmitted together. Antibodies to Chlamydia were detected by the indirect FA test in sera from all If men with gonococcal urethritis from whom Chlamydia was isolated. In the present test method we used a single antigen, SA2(f). The microimmunofluorescence test devised by Wang [14], which we have used in a previous serological study of NGU [10], makes use of at least 10 different antigens. The microimmunofluorescence test is useful in typing chlamydial isolates, and it has been shown that serotypes designated D and E are those predominantly associated with genital infections. As a test for screening large numbers of sera, however, the microimmunofluorescence test is laborious; therefore, we sought a single antigen that would detect antibodies to the predominant serotypes.
381
382
References
1. Holmes, K. K., Johnson, D. W., Floyd, T. M., Kvale, P.A. Studies of venereal disease. II. Observations on the incidence, etiology, and treatment of the postgonococcal urethritis syndrome. J. A. M. A. 202:467-473, 1967. 2. Boyd, J. T., Csonka, G. W., Oates, J. K. Epidemiology of non-specific urethritis. Br. J. Vener. Dis. 34:40-43, 1958. 1 J. D. Oriel, P. Reeve, and C. S. Nicol, "Minocycline in the. Treatment of Nonspecific Genital Infection," manuscript in preparation.
3. Masterton, G., Schofield, C. B. S. Doxycycline' HO (Vibramycin) as a single dose oral treatment of gonococcal and non-specific urethritis in men. Br. J. Vener. Dis. 48:121-125, 1972. 4. Dunlop, E. M. C., Vaughan-Jackson, J. D., Darougar, S., Jones, B. R. Chlamydial infection. Incidence in "non-specific" urethritis. Br. J. Vener. Dis. 48:425-428, 1972. 5. Oriel, J. D., Reeve, P., Powis, P., Miller, A., Nicol, e. S. Chlamydial infection. Isolation of Chlamydia from patients with non-specific genital infection. Br. J. Vener. Dis. 48:429-436, 1972. 6. Richmond, S. J., Hilton, A. L., Clarke, S. K. R. Chlamydial infection. Role of Chlamydia subgroup A in non-gonococcal and post-gonococcal urethritis. Br. J. Vener. Dis. 48:437-444, 1972. 7. Jawetz, E. Chemotherapy of chlamydial infections. Adv. Pharmacol. Chemother, 7:253-282, 1969. 8. Felarca, A. B., Laqui, E. M., Ibarra, L. M. Gentamicin in gonococcal urethritis of Filipino males: dosage and response. J. Infect. Dis. (Suppl.) 124:S287-S292, 1971. 9. Wentworth, B. B. Use of gentamicin in the isolation of subgroup A Chlamydia. Antimicrob. Agents Chemother. 3:698-702, 1973. 10. Reeve, P., Gerloff, R. K., Casper, E., Philip, R. N., Oriel, J. D., Powis, P. A. Serological studies on the role of Chlamydia in the aetiology of non-specific urethritis. Br. J. Vener. Dis. 50:136-139, 1974. II. Phillips, I., Humphrey, D., Middleton, A., Nicol, e. S. Diagnosis of gonorrhoea by culture on a selective medium containing vancomycin, colistin, nystatin and trimethoprim (VCNT). A comparison with gramstaining and immunofluorescence. Br. J. Vener. Dis. 48:287-292, 1972. 12. Wang, S. P., Grayston, J. T. Immunological relationship between genital TRIC, lymphogranuloma venereum and related organisms in a new microtiter indirect immunofluorescence test. Am. J. Ophthalmol. 70:367-374, 1970. 13. Lawrence, A. G., Phillips, I., Nicol, e. S. Gentamicin in sexually transmitted diseases. Postgrad. Med. J. 50(Suppl. 7):33-35, 1975. 14. Wang, S. P. A micro-immunofluorescence method. Study of antibody response to TRI C organisms in mice. In R. L. Nichols [ed.]. Trachoma and related disorders caused by chlamydial agents; proceedings of a symposium. Excerpta Medica, Amsterdam, 1971, p. 237-288. 15. Wang, S. P., Grayston, J. T. Classification of TRIC and related strains with microimmunofluorescence. In R. L. Nichols [ed.]. Trachoma and related disorders caused by chlamydial agents; proceedings of a symposium. Excerpta Medica, Amsterdam, 1971, p. 305-321. 16. Rodin, P. Asymptomatic non-specific urethritis. Br. J. Vener. Dis. 47:452-453, 1971. 17. Portnoy, J., Mendelson, J., Clecner, B. and Heisler, L. Asymptomatic gonorrhoea in the male. Can. Med. Assoc. J. 110:169-171, 1974.
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The accuracy of the stated history may be doubtful, either because of deliberate falsification by the patient or because of a previous asymptomatic attack of gonorrhoea or NGV, both of which are known to occur [16, 17]. In addition, there could have been a previous exposure to nongenital strains of Chlamydia. The significance of positive serological results in patients with gonococcal urethritis or N GU who do not yield Chlamydia on cell culture and who give no past history of urethritis requires further investigation. It seems probable to us that Chlamydia is one of the causes of PGV. In NGV, a disease which clinically resembles PGU, we have evidence that the use of a tetracycline simultaneously cures the urethritis and eliminates Chlamydia from Chlamydia-positive patients ,1 and while we have not studied the effect of treatment on Chlamydia associated with PG V, it is known that tetracyclines cure most cases of PGV. We do not suggest that Chlamydia is the only cause of PGV. There are many patients in whom Chlamydia cannot be demonstrated, and the causative organism or organisms of the urethritis in these patients have yet to be identified. A comparable situation exists in N G V, in which the cause of approximately half the infections is unknown. Nevertheless, in the present investigation, of 26 patients who developed P GV one to two weeks after treatment with gentamicin, 15 (58%) yielded Chlamydia on cell culture before treatment or during followup, and it seems to us likely that these organisms may playa significant part in the causation of the disease.
Oriel et al.
APRIL 1975
Infection with Chlamydia Group A in Men with Urethritis Due to Neisseria gonorrhoeae J. D. Oriel, P. Reeve, B. J. Thomas, and C. S. Nicol
From the Department of Venereology, St. Thomas' Hospital, and the Department of Bacteriology, University College Hospital Medical School, London, England
After treatment for urethritis caused by N eisseria gonorrhoeae, a proportion of male patients develop postgonococcal urethritis (PGU); their urethral discharge recurs and an excess of polymorphonuclear leukocytes is seen on microscopy of urethral material, but cultures for N. gonorrhoeae are negative. The possible causes of PGU have been discussed by Holmes et al. [1]. Although a few cases may be due to slow resolution of urethral inflammation or possibly to the emergence of gonococcal L-forms, it is thought that in the majority of men PGU is due to an associated nongonococcal urethritis (NGU), the two infections, gonorrhea and NGU, having been contracted at the same time. The longer incubation period of NGU [2] explains why PG U appears when the gonococcal infection is subsiding. The clinical features of
PGU and NGU are similar, and both respond to treatment with tetracyclines; furthermore, if gonorrhea is treated with a tetracycline, the incidence of PG U is significantly lower [3]. Thus it seems likely that the agent or agents which cause NGU may cause a proportion of PGU also. The etiology of N GU is not fully understood at pre sent. The urethritis is again evidenced by an excess of polymorphonuclear leukocytes seen on microscopy of urethral material and by the absence of N. gonorrhoeae. A few cases may be caused by bacterial infection secondary to urethral trauma or disease, or by trichomonal, candidal, or herpetic infections. But the majority of cases of N GU cannot be attributed to these causes and are thought to be due to the sexual transmission of other pathogenic organisms. There is now evidence that Chlamydia group A (Chlamydia trachomatis) is an important cause of N G U. By the use of a sensitive cell-culture technique, Chlamydia has been recovered from approximately 40% of men with N GU, and from many of their female sexual contacts [4, 51. On the other hand, attempted isolations of Chlamydia from control groups of men without urethritis have given positive results in less than 5% of cases [5, 6]. Chlamydia may also be related to PGU. In a
Received for publication June 24, 1974, and in revised form December 4, 1974. We are grateful to Mr. J. M. McCaUig for technical assistance with serology. Gentamicin was kindly supplied by Roussel Laboratories, Ltd., Wembly Park, Middlesex, England. This work was supported by grants from the Medical Research Council (Great Britain). Please address requests for reprints to Dr. J. D. Oriel, Department of Venereology, S1. Thomas' Hospital, London, S.E. 1, England.
376
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Men with urethritis due to Neisseria gonorrhoeae were treated with gentamicin, which is inactive against Chlamydia. Urethral specimens were collected before treatment and one and two weeks after treatment and cultured for Chlamydia in irradiated McCoy cells. The overall incidence of chlamydial infection was 15 of 44 (34%). All of 15 Chlamydia-positive men and 11 of 29 Chlamydia-negative men (38%) developed postgonococcal urethritis two weeks after treatment. Pretreatment sera were examined by a complement fixation test and a simplified indirect fluorescent antibody (FA) test with a yolk sac-cultured antigen, strain SA2(t). Sera from all of 15 Chlamydia-positive men had titers of ~1:16 in the indirect FA test; 13 of 29 Chlamydia-negative men (45%) had positive tests. The complement fixation test was insensitive, detecting chlamydial antibodies in only one of 15 sera from Chlamydia-positive men. These results suggest that Chlamydia may cause many cases of postgonococcal urethritis.
Chlamydia in Gonococcal Urethritis
Materials and Methods
All the patients were seen in the Department of Venereology, St. Thomas' Hospital, London, between February 1 and April 30, 1973. On their first attendance at the clinic, after general and local examinations, men provided specimens as follows. A specimen of urethral secretion was taken with a wire loop and spread on a slide for immediate gram-staining and microscopy. A second urethral specimen was inoculated onto a culture plate for detection of N. gonorrhoeae. A third specimen was mixed on a slide with a drop of saline and immediately examined for Trichomonas vaginalis . The remaining urethral material was collected with a
cotton-wool meatal swab that was broken into a 2-ml screw-capped bottle of cell culture medium for culture of Chlamydia. Urine was passed into two glasses. Blood was collected for serology for syphilis and Chlamydia. When the diagnosis of gonococcal urethritis had been made, the patient was immediately treated with a single im injection of gentamicin (240 mg), Consent to the use of this drug was obtained after the patient had been warned that there was a slight risk of toxic reaction, although in fact none was experienced. The patient was advised to abstain from sexual intercourse and the consumption of alcohol for at least two weeks. Follow-up examinations were performed after two to three days, seven days, and 14 days. Specimens were normally collected at least 2 hr after micturition and were examined as on the patient's first attendance, but cultures for Chlamydia were repeated only on the seventh and 14th days after treatment. Assessment of PGU. The diagnosis of POD was made if there was evidence of significant urethritis more than seven days after treatment. Although many men with PGU had symptoms such as dysuria or urethral discharge, we did not rely on clinical symptoms or signs in making the diagnosis. Significant urethritis was defined in terms of the numbers of polymorphonuclear leukocytes per mean high-power field (x 100 objective) on a gram-stained urethral smear; occasionally a centrifuged specimen of first-catch urine was used similarly. A patient who showed more than 20 polymorphonuclear leukocytes per mean high-power field was considered to have significant urethritis. Laboratory methods. Culture for N. gonorrhoeae was performed on a selective medium [11J. Cell culture for Chlamydia group A was undertaken by centrifugation of clinical specimens onto irradiated McCoy cells, followed by incubation and examination for chlamydial inclusions; this method has been described [5]. Two serological tests were used to detect chlamydial antibodies: C F test with a microtiter technique and a Chlamydia group-reactive yolk-sac antigen, and a simplified indirect FA test, modified from the method of Wang and Grayston [12]. A yolk sac-cultured antigen, strain SA2 (0, was used. This was partially
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recent investigation Chlamydia was recovered from 29% of a group of men with gonococcal urethritis, and it was reported that after treatment with penicillin the incidence of PG U was higher in Chlamydia-positive than in Chlamydia-negative men [6]. However, penicillin inhibits the growth of Chlamydia [7], a fact that limits the value of studies of the relationships between Chlamydia and PG U in men treated with this drug. On the other hand, gentamicin, which is an effective treatment for gonorrhea [8], is known to have no in vitro action against Chlamydia [9]. In the present investigation, the relationship between Chlamydia and PGU has been studied in a group of men with gonococcal urethritis who were treated with gentamicin. Urethral specimens for cell culture for Chlamydia were collected before treatment and again one and two weeks after treatment, and the incidence of POD was compared in Chlamydia-positive and Chlamydia-negative patients. It is known that there is a serological response to genital chlamydial infection; antibodies have been detected by CF tests, FA techniques, and a radioisotope precipitation test [10]. During the present investigation, the first two of these methods were used to detect antibodies to Chlamydia in sera collected from the men with gonococcal urethritis on their first attendance in a clinic. Results of the serological tests were then compared with the results of isolation of Chlamydia.
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Oriel et al.
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Results
Initially, 82 heterosexual men with confirmed gonococcal urethritis were investigated, but 38 were removed from the project for the following Table 1.
reasons: default, 16; initial contaminated specimen, eight; relapse of gonococcal urethritis after treatment with gentamicin, necessitating retreatment with another antibiotic, 9; reinfection with N. gonorrhoeae during follow-up, two; sera not available, three. This left 44 patients available for further study. Effect of repeated specimen collection on isolations of Chlamydia. Of the 44 patients studied completely, 11 (25%) yielded Chlamydia from urethral specimens collected on their first attendance. Of these 11 men, nine remained Chlamydia-positive after one and two weeks; one man was still positive after one week but became negative after two weeks; and one man, having still been positive after one week, gave a contaminated specimen after two weeks. However, four men who were initially Chlamydianegative yielded isolates subsequently, three of them after one week and one after two weeks (table 1). If the patients who yielded isolates at any time during the two-week study period are included, the incidence of urethral Chlamydia is raised to 15 of 44 men with gonococcal urethritis (34%).
Men with gonococcal urethritis who yielded isolates of Chlamydia on cell culture. First attendance
Patient no. I 2
History
Duration of symptoms (days)
G
]
G
A
One week after treatment
Chlam.
VD
PGV
Chlam.
+ +
+
+ + +
+ + + + + + + + + + +
3
2
4 5 6 7 8*
2 4 2 7
+ +
A
+ + + +
NGV
2]
9t lOt
21
]I
28
12
2
13
2 7
14 15
2
Two weeks after treatment
+ + +
+ + + + + + + +
+ + + + + + + +
+ +
+ + +
vn
PGV
Chlam.
+ +
+ + +
+ +
+
+ + +
+
+ + + + + + + + + + +
+ + + + + +
NOTE. A = asymptomatic; C = contaminated specimen; Chlam. = culture for Chlamydia; 0 nongonococcal urethritis; PO V = postgonococcal urethritis; V D = urethral discharge. * Patient also had trichomoniasis; he was treated with metronidazole. t· Patient had intercourse during follow-up.
=
C
+ C
+ + + + +
gonorrhea; NOV =
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purified by density gradient centrifugation and applied to Teflon-coated slides, dried, and fixed in acetone. This antigen was allowed to interact with serial twofold dilutions of test sera for 30 min at 37 C in a moist chamber, and then washed for 10 min with two changes of phosphate-buffered saline to remove uncombined serum. Fluorescein-conjugated antihuman globulin (Wellcome fluorescent antibodies, anti-human immunoglobulin-sheep, Wellcome Laboratories, London) was then applied to the preparation and allowed to react for 30 min at 37 C in a moist chamber; this procedure permitted detection of antigen-bound globulins. Fluorescence was observed with a Gillett and Sibert blue light fluorescence microscope. Phosphate-buffered saline was used as a diluent throughout. The serum titer was recorded as the highest dilution at which particulate fluorescence could be detected.
379
Chlamydia in Gonococcal Urethritis
Table 2.
Men with gonococcal urethritis who did not yield isolates of Chlamydia on cell culture. First attendance
Patient no.
16 17 18 19 20 21 22* 23 24 25t 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42t 43 44
History
Duration of symptoms (days)
G G,NGU G,NGU G,NGU NGU
3 2 1 3 1 5
G,NGU G G,NGU G,NGU G,NGU G,NGU NGU
A
NGU
Chlam.
UD
PGU
Chlam.
+ + +
C
+
UD
PGU
+
+ +
+
+
+ +
+
+
+
A
+ +
+ +
+
+
+ +
C
+
A
2
Chlam.
C
4 1 7 1 3 2 1 4
Two weeks after treatment
One week after treatment
C
1
1 5 3 14 3 6 3
+
+ C
+ +
+ +
C
C
2
NGU G,NGU
A I
+
+
NOTE. A = asymptomatic; C = contaminated specimen; Chlam. = culture for Chlamydia; G nongonococcal urethritis; PG U = postgonococcal urethritis; UD = urethral discharge. * Patient did not have PG U three weeks after treatment. t Patient had intercourse during follow-up.
=
gonorrhea; NGU
=
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Chlamydia isolations and PGU. Of the 15 men who gave chlamydia isolates on the first, second, or third attempt, significant urethritis (indicating PG U) was present one week after treatment in 13 patients and two weeks after treatment in all 15. After two weeks, nine of the 15 men with PGU had a manifest urethral discharge; the remaining six men had no discharge. Of the 29 men who were consistently Chlamydia-negative, significant urethritis (indicating PG U) was present one week after treatment in 12 and two weeks after treatment in 11 (38%). Of these 11 men, four had a manifest
Isolation of Chlamydia and duration of symptoms. The duration of symptoms (urethral discharge and dysuria) in the 15 men with gonococcal urethritis who yielded chlamydia isolates was one to 28 days (mean, 7.76 days; SD, 8.99); in the 29 men with gonococcal urethritis who were consistently Chlamydianegative, however, the duration of symptoms was one to 14 days (mean, 3.16 days; SD, 2.69) (table 2). This suggests that in men with gonococcal urethritis the duration of symptoms may affect the likelihood of isolation of Chlamydia.
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380
Discussion
Chlamydia can often be recovered from the urethra of men with gonococcal urethritis. In a previous investigation we have found that of 104 valid urethral specimens from these men, collected on their first attendance, 22 (21 %) yielded Chlamydia in cell culture (authors' unpublished observations). This figure compares with the 25% rate of isolation found in the present study and with the results reported by other workers [6].
Not all the Chlamydia-positive men yielded isolates from the first urethral specimen. Little is known of the effect of repeated attempts at isolation on the isolation rate in either gonococTable 3.
cal urethritis or N GU, because patients with these diseases are normally treated on their first attendance with an antibiotic that may inhibit Chlamydia. There are two possible explanations for our results. Either our cell-culture system was not uniformly sensitive, or the chances of isolating Chlamydia from the urethra were increased by the passage of time, as the number of infective particles increases. The cell culture system did not appear to be insensitive, since we found that once patients had become Chlamydia-positive they usually remained so in subsequent specimens. We believe that the second supposition is more likely to be correct. This belief is strengthened by the observation that the mean duration of symptoms in the Chlamydia-positive group was double that in the Chlamydianegative group. A relationship between the isolation of Chlamydia from the urethra and the duration of symptoms in both gonococcal urethritis and N G U has been shown [6]. In investigating a possible connection between Chlamydia and PGU it is important that the gonococcal infection is treated with an antibiotic that has no action against Chlamydia. The choice is very limited, and gentamicin appears to be the most suitable drug. Not only is it reasonably effective for the treatment of gonococcal urethritis, curing about 90% of patients [13], but it has been shown to have no effect on Chlamydia in vitro; indeed, Wentworth has recommended its introduction into transport and growth media for Chlamydia to reduce bacterial contamination [9]. We have found that in our patients with
Antibodies to Chlamydia and incidence of postgonococcal urethritis (PG U).
Developing PGU (no. sera tested)
Indirect fluorescent antibody test positive*
Isolation of Chlamydia: result (no.)
No. (%)
GMT
History of GU or NGU
Positive (15)
15(100)
27
3
Negative (11) Positive (0)
2(18)
32
2
Negative (18)
11(61)
23
4
Yes (26)
No (18) NOTE.
GMT = geometric mean titer; GU = gonococcal urethritis; NGU = nongonococcal urethritis.
* Serum titers of
~1:16.
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urethral discharge. All patients with PGU were treated with a course of tetracycline, but were not studied further in the present investigation. Serology. PGU was diagnosed in a total of 26 men, of whom 15 had yielded chlamydial isolates on cell culture. All of these 15 gave positive results with the indirect FA test at titers of ~1:16, the geometric mean titer (GMT) was 1:27, and three of the men gave a past history of urethritis. None of the 18 men who did not develop PGU yielded chlamydia isolates; eleven of the 18, however, were seropusitive at titers of ~ 1:16, and four of these gave a past history of urethritis (table 3). In contrast with the indirect FA test, the CF test was insensitive. Only one of 15 sera from patients who had given chlamydia isolates on cell culture gave a positive result with the CF test.
Chlamydia in Gonococcal Urethritis
The antigen SA2(f), which is indistinguishable from the L GV II strain by immunotyping, has been shown by FA tests to give a one-way cross-reaction with antisera to Chlamydia group A, including serotypes D and E. This reaction, together with its ease of culture, led us to select S A2(f) as antigen for the indirect FA test. We are aware of the possible limitations imposed by this choice. As Wang and Grayston pointed out [15], our antigen is of "senior" type, capable of detecting antibodies to the B, D-E, and LGV immunotype complex; it may not detect antibodies to another commonly found genital serotype, the F-G complex, and probably would not react with antibodies to the more distantly related C, H, I, and K immunotypes. However, in our previous study [10], in which serotypes F and I were included as antigens, no sera were found that reacted solely with these antigens without also crossreacting with the LGV II immunotype complex. We have no information about the reactions of antibodies to the serotypes G, H, and the most recently discovered K. In practice, SA2(f) is apparently an efficient single antigen for measuring chlamydial antibodies in sera from men with NGU and PGU, although we would accept the possibility that this simplified system may fail to detect antibodies to some of the rarer serotypes. The serological results of the present investigation agree with those that we obtained from study of men with Chlamydia-positive NO U, 86% of whom gave positive results with the microimmunofluorescence test [10]. In both gonococcal urethritis and N G U, the great majority of patients who yield Chlamydia on cell culture give evidence of the presence of chlamydial antibodies in the serum. However, in both diseases a proportion of men who did not yield chlamydial isolates had antibodies to the predominant chlamydia serotypes. Of 29 sera from patients with gonococcal urethritis from whom Chlamydia was not isolated, 14 (48%) gave positive results with the indirect FA test. Some of these men gave a history of gonococcal urethritis or N G U and could thus have been exposed to genital chlamydial infection in the past. Others gave no such history, and we can only speculate on the significance of the positive serological results in these patients.
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gonococcal urethritis who have been treated with gentamicin, PGU regularly developed in those who were Chlamydia-positive before treatment. All of the 11 men who were Chlamydia-positive before treatment later developed POU. Similarly, four men who were initially Chlamydia-negative but who later gave isolates also developed POU. On the other hand, only 11 of 29 men (38%) who were consistently Chlamydia-negative developed POD after treatment. A relationship appears to exist between urethral Chlamydia and PGU. Two questions may now be asked. First, was Chlamydia sexually transmitted at the same time as N. gonorrhoeae, or was Chlamydia already present in the urethra and in some way activated by the gonococcal infection? Second, does Chlamydia cause PG U in these patients or is it merely associated with it, the true cause lying elsewhere? The first question cannot be answered completely until more is known of the epidemiology of chlamydial infection associated with gonorrhoea. We are studying this in a group of men with gonococcal urethritis and their sexual contacts. Although it has been argued that infection with N. gonorrhoeae may activate a latent chlamydial infection in the male urethra [6], our own studies have indicated that Chlamydia is not demonstrable in the urethra in the absence of urethritis [5], and we think it is more likely that N. gonorrhoeae and Chlamydia are sexually transmitted together. Antibodies to Chlamydia were detected by the indirect FA test in sera from all If men with gonococcal urethritis from whom Chlamydia was isolated. In the present test method we used a single antigen, SA2(f). The microimmunofluorescence test devised by Wang [14], which we have used in a previous serological study of NGU [10], makes use of at least 10 different antigens. The microimmunofluorescence test is useful in typing chlamydial isolates, and it has been shown that serotypes designated D and E are those predominantly associated with genital infections. As a test for screening large numbers of sera, however, the microimmunofluorescence test is laborious; therefore, we sought a single antigen that would detect antibodies to the predominant serotypes.
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References
1. Holmes, K. K., Johnson, D. W., Floyd, T. M., Kvale, P.A. Studies of venereal disease. II. Observations on the incidence, etiology, and treatment of the postgonococcal urethritis syndrome. J. A. M. A. 202:467-473, 1967. 2. Boyd, J. T., Csonka, G. W., Oates, J. K. Epidemiology of non-specific urethritis. Br. J. Vener. Dis. 34:40-43, 1958. 1 J. D. Oriel, P. Reeve, and C. S. Nicol, "Minocycline in the. Treatment of Nonspecific Genital Infection," manuscript in preparation.
3. Masterton, G., Schofield, C. B. S. Doxycycline' HO (Vibramycin) as a single dose oral treatment of gonococcal and non-specific urethritis in men. Br. J. Vener. Dis. 48:121-125, 1972. 4. Dunlop, E. M. C., Vaughan-Jackson, J. D., Darougar, S., Jones, B. R. Chlamydial infection. Incidence in "non-specific" urethritis. Br. J. Vener. Dis. 48:425-428, 1972. 5. Oriel, J. D., Reeve, P., Powis, P., Miller, A., Nicol, e. S. Chlamydial infection. Isolation of Chlamydia from patients with non-specific genital infection. Br. J. Vener. Dis. 48:429-436, 1972. 6. Richmond, S. J., Hilton, A. L., Clarke, S. K. R. Chlamydial infection. Role of Chlamydia subgroup A in non-gonococcal and post-gonococcal urethritis. Br. J. Vener. Dis. 48:437-444, 1972. 7. Jawetz, E. Chemotherapy of chlamydial infections. Adv. Pharmacol. Chemother, 7:253-282, 1969. 8. Felarca, A. B., Laqui, E. M., Ibarra, L. M. Gentamicin in gonococcal urethritis of Filipino males: dosage and response. J. Infect. Dis. (Suppl.) 124:S287-S292, 1971. 9. Wentworth, B. B. Use of gentamicin in the isolation of subgroup A Chlamydia. Antimicrob. Agents Chemother. 3:698-702, 1973. 10. Reeve, P., Gerloff, R. K., Casper, E., Philip, R. N., Oriel, J. D., Powis, P. A. Serological studies on the role of Chlamydia in the aetiology of non-specific urethritis. Br. J. Vener. Dis. 50:136-139, 1974. II. Phillips, I., Humphrey, D., Middleton, A., Nicol, e. S. Diagnosis of gonorrhoea by culture on a selective medium containing vancomycin, colistin, nystatin and trimethoprim (VCNT). A comparison with gramstaining and immunofluorescence. Br. J. Vener. Dis. 48:287-292, 1972. 12. Wang, S. P., Grayston, J. T. Immunological relationship between genital TRIC, lymphogranuloma venereum and related organisms in a new microtiter indirect immunofluorescence test. Am. J. Ophthalmol. 70:367-374, 1970. 13. Lawrence, A. G., Phillips, I., Nicol, e. S. Gentamicin in sexually transmitted diseases. Postgrad. Med. J. 50(Suppl. 7):33-35, 1975. 14. Wang, S. P. A micro-immunofluorescence method. Study of antibody response to TRI C organisms in mice. In R. L. Nichols [ed.]. Trachoma and related disorders caused by chlamydial agents; proceedings of a symposium. Excerpta Medica, Amsterdam, 1971, p. 237-288. 15. Wang, S. P., Grayston, J. T. Classification of TRIC and related strains with microimmunofluorescence. In R. L. Nichols [ed.]. Trachoma and related disorders caused by chlamydial agents; proceedings of a symposium. Excerpta Medica, Amsterdam, 1971, p. 305-321. 16. Rodin, P. Asymptomatic non-specific urethritis. Br. J. Vener. Dis. 47:452-453, 1971. 17. Portnoy, J., Mendelson, J., Clecner, B. and Heisler, L. Asymptomatic gonorrhoea in the male. Can. Med. Assoc. J. 110:169-171, 1974.
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The accuracy of the stated history may be doubtful, either because of deliberate falsification by the patient or because of a previous asymptomatic attack of gonorrhoea or NGV, both of which are known to occur [16, 17]. In addition, there could have been a previous exposure to nongenital strains of Chlamydia. The significance of positive serological results in patients with gonococcal urethritis or N GU who do not yield Chlamydia on cell culture and who give no past history of urethritis requires further investigation. It seems probable to us that Chlamydia is one of the causes of PGV. In NGV, a disease which clinically resembles PGU, we have evidence that the use of a tetracycline simultaneously cures the urethritis and eliminates Chlamydia from Chlamydia-positive patients ,1 and while we have not studied the effect of treatment on Chlamydia associated with PG V, it is known that tetracyclines cure most cases of PGV. We do not suggest that Chlamydia is the only cause of PGV. There are many patients in whom Chlamydia cannot be demonstrated, and the causative organism or organisms of the urethritis in these patients have yet to be identified. A comparable situation exists in N G V, in which the cause of approximately half the infections is unknown. Nevertheless, in the present investigation, of 26 patients who developed P GV one to two weeks after treatment with gentamicin, 15 (58%) yielded Chlamydia on cell culture before treatment or during followup, and it seems to us likely that these organisms may playa significant part in the causation of the disease.
Oriel et al.
