THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.4. OCTOBER 1978 © 1978 by The University of Chicago. 0022-1899/78/3804-0003$00.95
Diagnosis and Etiology of Nongonococcal Urethritis Stephen L. Swartz, Stephen J. Kraus, Kenneth L. Herrmann, Michael D. Stargel, W. Jerry Brown, and Stephen D. Allen
From the Venereal Disease Research Branch and the Enterobacteriology Branch, Bacteriology Division, and the Perinatal Virology Branch, Virology Division, Center fOT Disease Control, Atlanta, Georgia
Nongonococcal urethritis (NGU) presently accounts for more than half of the cases of urethritis seen in sexually active males attending venereal disease clinics in the United States [1, 2]. Diagnosis of NGU is currently one of exclusion [3], the therapy is controversial [4, 5], and the incubation period [6] and management of sexual partners are uncertain [7]. Clinical studies and management of patients with NGU would be aided if NGU were more clearly defined and its etiology were better understood [8]. Inadequate criteria are available to differentiate patients with minimal symptomatology of urethritis from patients with "psychosomatic urethritis" who have no objective evidence of urethral inflammation on
physical and laboratory examination. One objective of this study was to determine the minimal number of polymorphonuclear cells (PMN) that must be present in a gram-stained smear of urethral secretion to distinguish physiologic urethral exudate from pathologic urethral discharge. Previous attempts to determine the etiology of NGU have focused on Chlamydia trachomaiis,
Mycoplasma hominis, Ureaplasma urealsticum, Herpesvirus hominis, and Trichomonas vaginale. At present, only C. trachomatis and possibly U. urealyticum have been implicated as potential causes of NGU in appropriately controlled studies. The second objective of this study was to examine the occurrence of other microorganisms in patients with NGU.
Received for publication June 20, 1977. and in revised form April 13, 1978. We thank Dr. Donald G. Ahearn, Georgia State University, for yeast isolation and identification; Jay S. Smith, Statistical Activities Branch, Center for Disease Control, for statistical assistance; and Pamela Routh, Lillian Cooper, and Rex Poole of the DeKalb County Venereal Disease Clinicfor their assistance. Useof trade names is for identification only and does not constitute endorsement by the U.S. Public Health Service or by the U.S. Department of Health, Education,and Welfare. Please address requests for reprints to the Center for Disease Control. Technical Information Services, Bureau of StateServices, Atlanta, Georgia 30333.
Materials and Methods
Study population. The study population included 280 men attending the DeKalb County Venereal Disease Clinic, Atlanta, Ga., from October 1975 through March 1976. Clinic patients were excluded if they had received any antibiotics within the preceding four weeks, if they had a visible deformity of the urethral meatus on physical examination, or if they failed to give in-
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The observation of more than four polymorphonuclear cells (PMN) per high-power field (hpf) in gram-stained smears of urethral secretions was found to differentiate patients with urethritis from patients without urethritis. A urethral discharge was present in 78% of patients with nongonococcal urethritis (NGU). Dysuria without demonstrable urethral discharge and with fewer than four PMN /hpf did not appear to fit into the NGU spectrum. NGU is now defined to include men who have negative urethral cultures for Neisseria gonorrhoeae with a urethral discharge and/or more than four PMN /hpf in their urethral smears. The finding of more than four PMN /hpf in the urethral smears of 22% of asymptomatic sexually active men with more than one sexual partner (polygamous controls) suggeststhat asymptomatic NGU is not uncommon. Chlamydia trachoma tis was isolated significantly more frequently from the NGU study group than from the control group (P < 0.001). This study adds Corynebacterium vaginale (Haemophilus vaginalis) , group B streptococci, and yeasts to the list of sexually transmitted microorganisms that are not etiologic determinants of NGU.
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=
Hewitt broth for subsequent culture of group B streptococci. A second swab was inserted I em further into the anterior urethra, inoculated directly onto VCN medium (Thayer-Martin media without hemoglobin) for culture of Neisseria gonorrhoeae, and then placed in sucrose-phosphate transport media for C. trachomatis. A third swab was obtained in the same manner, inoculated directly onto Sabouraud's media for yeasts, and then placed in I ml of Eagle's minimal essential medium (MEM) for viral studies. After reviewing the results from the initial 100 samples, we decided that the incidence of fungal and viral isolation was too low to justify analyzing the third swab for such organisms, and for the rest of the study, these swabs were directly inoculated onto prereduced anaerobic media and immediately placed in an oxygen-free environment. Each 2- X l-cm smear of urethral secretion was gram-stained and examined for the presence of PMN and gram-negative diplococci. The slide was first scanned under low power (x 100) to locate areas with the highest concentration of PMN. These areas were then examined under high magnification (x970), and the number of PMN in the five microscopic fields with the highest number of PMN were counted and averaged. Preliminary studies have shown that while the total number of PMN in many areas on a slide averaging fewer than four PMN jhpf may outnumber the total number of PMN on a slide averaging four or more PMN jhpf, only rarely «1.5% of slides examined) does this situation occur. Consequently, this finding need not be considered a problem in our laboratory evaluation of urethral specimens. Identification of microorganisms by culture. C. vaginale. The PSD medium [10, II] was modified by the addition of I ml of IsoVitalex" (Baltimore Biological Laboratories [BBL], Cockeysville, Md.)jI00 ml of medium, an addition which facilitated growth of certain isolates of C. vaginale that grew poorly on standard PSD media. After the plates were inoculated with the urethral sample, they were incubated at 36.5 C for 48 hr in a candle-extinction jar. C. vaginale was identified on the basis of (1) colonial morphology as seen at a magnification of x40 with a stereoscopic microscope [II]; (2) inhibition of
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formed written consent. All other male patients were prospective candidates since this study included men with gonococcal urethritis (GU), men with NGU, and men without urethritis who were sexually active and had more than one sexual partner (polygamous). The last group included men who were seen at the clinic either for routine examination or for evaluation of external genital lesions such as condylomata acuminata, scabies, pediculosis pubis, or syphilis. The study also included a monogamous control group consisting of 25 male volunteers who were matched by age to the clinic population. Each of these volunteers (1) had had sexual intercourse with only one person during the preceding 90 days, (2) was free of symptoms of urethritis, and (3) had no urethral discharge on physical examination. Patients with gonorrhea received either aqueous procaine pencillin G plus probenecid or tetracycline hydrochloride according to the recommendations of the U.S. Public Health Service [9], and men with NGU were treated with 500 mg of tetracycline hydrochloride four times a day for seven days. Evaluation and collection of urethral specimens. All patients were interviewed and examined by the same physician (S.L.S.). Any urethral discharge was noted and quantitated as follows: 0 = no discharge present after penile stripping (i.e., compressing the urethra from the bulb distally); 1+ = no spontaneous discharge, but slight discharge present after penile stripping; 2+ = no spontaneous discharge, but moderate discharge present after penile stripping; 3+ spontaneous discharge present. Three urethral specimens were obtained in random sequence by insertion of calcium alginate swabs into the urethra. A preliminary study of NGU had shown that if each swab was inserted -I em further into the urethra, the numbers of PMN per high-power field (hpf) from three consecutive swabs were not significantly different. A gram stain was prepared from each swab by rolling the swab once over a 2- X l-cm area on a glass slide to achieve as uniform a dispersion of PMN over the slide as possible; the swab was then inoculated onto a plate of modified PSD media (protease peptone, starch, and dextrose) for culture of Corynebacterium vaginale (Haemophilus vaginalis) and placed in 5 ml of Todd-
Nongonococcal Urethritis
blast cells. The culture tubes were transported to the laboratory within 6 hr of inoculation and incubated at 37 C [24]. The cultures were observed daily for one week for evidence of CPE characteristic of H. hominis infection and then weekly for one month for evidence of CPE characteristic of CMV infection. H. hominis isolates were typed with use of indirect HAl [25]. A naerobes. All urethral swabs to be analyzed for anaerobic organisms were inoculated immediately in the clinic onto one plate of blood agar (5% rabbit blood in trypticase soy agar containing yeast extract, vitamin K1, and hemin) [26], one plate of the same medium modified by the addition of neomycin (final concentration, 100 g/ml), and a tube of thioglycolate broth (BBL 0135C) supplemented with 10 g of heminjrnl and 10 g of vitamin K1/ml. The media were prereduced for at least 18 hr in an atmosphere of 85% N 2 , 10% H 2 , and 5% CO 2 before use. Exposure to oxygen during inoculation was minimized by a holding-jar procedure modified from that of Martin [27] by use of a mixture of 85% N 2 , 10% H 2 , and 5% CO 2 passed through a palladium catalyst and by use of the holding jars at the time of primary inoculation in the clinic. Following inoculation, all plates and tubes were incubated for five days at 35 C in GasPak jars with disposable GasPak H 2-C0 2 generators (BBL). After incubation each plate was carefully examined under a dissecting microscope, and with the aid of a modified holding-jar technique, one or more representatives of each colony type were picked (usually four to eight colonies per plate) for anaerobic subculture and purification on blood agar. The pure-culture isolates were characterized by colonial and gram-stain morphology, presence or absence of endospores, aerotolerance on blood agar plates, and gas-liquid chromatographic analysis of metabolic products in Schaedler broth (BBL) culture (4872 hr) using the procedures of Dowell and Hawkins [28]. The identification of genera reported here was only presumptive and was based on the data of Dowell and Hawkins [28]. A X2 test was applied to all contingency tables [29] to check for the degree of independence among the variables of interest. The level of significance was chosen to be 5% in all cases. Yates's
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growth by 3.0% H 202 [11]; (3) a negative catalase reaction [11]; (4) a gram stain showing groups of variably staining small coccoids and short pleomorphic rods [12]; (5) incomplete phemolysis of freshly prepared rabbit blood agar [13]; and (6) fermentation of dextrose, maltose, and starch, but no fermentation of mannitol or rhamnose [13]. All C. vaginale isolates were confirmed by Dr. W. E. Dunkelberg, Medical Laboratory Detachment, Dwight David Eisenhower Army Medical Center, Fort McPherson, Ga. Group B streptococci. From the test tube containing 5 ml of Todd-Hewitt broth, pour and streak plates were made for isolation of pure colonies of group B streptococci [I4]. Pure cultures of these isolated colonies were extracted by the hot HCl method of Swift et al. [I5]. The extracted streptococcal antigen was added to specific grouping and typing antisera in sterile capillary tubes, and the appearance of a precipitin reaction was noted [16]. N. gonorrhoeae. Gonococci in specimens inoculated onto VCN medium [I7] were identified by standard techniques [I8, 19]. C. trachomatis. The sucrose-phosphate transport tubes were kept frozen at -70 C until the time of inoculation. McCoy cells, treated with 5iodo-2'-deoxyuridine according to the technique of Wentworth and Alexander [20], were grown as monolayers on cover slips in I-dram vials. The clinical specimens were inoculated onto these cover slips, centrifuged at 3,000 g at 35 C for 60 min [21], and incubated for three days. The cover slips were fixed in methanol, stained with Jones-iodine solution [20], and examined at X 400 magnification for typical intracytoplasmic inclusions. Yeasts. Mycological agar (Difco, Detroit, Mich.) fortified with 0.5 g of chloramphenicol was used for the primary isolation of yeasts. All isolates were screened by the germ-tube test for the presence of Candida albicans [22]. A germ tube-negative isolate and a germ tube-positive isolate were speciated with the Uni-Yeast-Tek system (Corning Medical, Corning, N.Y.) [23]. Viruses. Specimens for isolation of H. hominis and cytomegalovirus (CMV) were placed immediately onto I ml of Eagle's MEM for 15-30 min and then inoculated directly onto rollertube cultures of human embryonic lung fibro-
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correction was used where cell sizes were fewer than five patients in two-way tables [30]. Fisher's exact probability is not given because of the number of observations available. Results
Numbers of PMN in urethral specimens. The mean number of PMN jhpf was determined for each of 299 slides. Of the 61 patients with a diagnosis of gonorrhea (gonorrhea being defined as urethritis from which N. gonorrhoeae was cultured), all but one had ee 50 PMN jhpf and typical gram-negative diplococci usually located intracellularly but occasionally only extracellularly. The 25 monogamous controls were without discharge or dysuria and had an average count (±SE) of 0.8 ± 0.7 PMN jhpf (range, 0-2.2). A histogram describing the PMN counts in urethral smears for polygamous men without discharge or dysuria and for NGU patients (with both discharge and dysuria) is presented in figure 1. Although some NGU patients had large 80
1
78:1 26 24
o
22
I
20 c
..,
..
18
0. 0
16
0
Polygamous Men without Dysuria or Discharge
NGU with Dysuria and Discharge
:;
e,
'0
*
14 12 10 8 6 4
rI
2 l-
4
8
12
16
20
24
28
32
36
40
I II 44
48
52
56
60
64
I
68
72
76
80
84
88
92
96
100
No. of PMN/hpf
Figure 1. Histogram of counts of polymorphonuclear cells (PMN) per high-power field (hpf) in urethral smears from patients with nongonococcal urethritis (NGU) with dysuria and discharge and from men without dysuria or discharge who were sexually active and had more than one sexual partner (polygamous control group).
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numbers of urethral PMN, an overlap in the number of PMN /hpf existed among many other NGU patients and the polygamous men without signs or symptoms of urethritis. For determination of the number of PMN jhpf that would best separate patients into these two groups, a table was constructed in which the counts of PMN jhpf for both groups are given in consecutive integers as cutoff points (table 1). To obtain the highest degree of sensitivity and specificity in choosing a PMN count that separates patients with urethritis from those without urethritis, we selected the row showing the minimal percentages of false-negative results and false-positive results (i.e., where the difference between these values was the smallest). This row corresponded to a count of four PMN jhpf. When a histogram for the 31 patients with urethral discharge alone was constructed, 29% had fewer than four PMN. This false-negative rate is similar to that of 23.8% obtained for "typical" NGU patients with both discharge and dysuria. However, when the histogram for the 26 patients
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of the patients. Among the 100 patients studied for the presence of anaerobic organisms, no significant differences in overall rates of isolation were noted for the GU group, the NGU study group, and the polygamous and monogamous control groups (P = 0.575) (table 2). An average of seven different anaerobic genera were isolated from each of these groups. Comparison of isolation rates for individual anaerobic organisms revealed statistically higher isolation rates for Propionibacterium-Arachnia (P < 0.001) and lower isolation rates for peptostreptococci (P = 0.024) in the combined monogamous and polygamous control groups than in the combined NGU and GU groups. Group B streptococci and C. vaginale were isolated from the NGU, GU, and polygamous control patients at statistically comparable rates (P = 0.670 and 0.705, respectively) (table 3). For the group B streptococci, the isolation rates for all serotypes were similar within each of the patient populations, with serotype III always the most prevalent. C. trachomatis was isolated significantly more frequently from the NGU study group than from the polygamous control group (P < 0.001) (table3). Analysis of the isolation rates of group B streptococci and C. vaginale in C. trachomatis-positive
Table 1. Counts of polymorphonuclear cells (PMN) in urethral smears from patients with nongonococcal urethritis (NGU) having dysuria and urethral discharge and from men without dysuria or discharge who were sexually active and had more than one sexual partner (polygamous control group).
Cutoff count ofPMN 1
2 3 4 5 6 7 8 9 10 11 12 13
14 15 16
NGo patients (percentage of false-negative results) 15.2 17.4 21.7 23.8 28.1 30.3 32.3 32.3 34.4 43.1 47.4 47.4
49.5 53.8 53.8 53.8
Percentage of false-positive results (patients with asymptomatic NGU)* 37.2 27.7 23.8 22.0 19.3 18.2 17.2 16.3 12.5 10.6 8.6 7.8 5.8 5.0 4.0 1.2
*Calculated by 100% minus the value for polygamous men without dysuria or discharge.
Polygamous men without dysuria or discharge (cumulative percentages) 62.8 72.3 76.2 78.0 80.7 81.8 82.8 83.7 87.5 89.4 91.4
92.2 94.2 95.0 %.6 93.8
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with dysuria alone was analyzed, 76.9% had fewer than four PMN. This value is similar to the 78% of polygamous men without discharge or dysuria who had fewer than four PMN and suggests that dysuria alone cannot be used to place patients in the NGU group. Recognizing that a definition of NGU based solely on a count of more than four PMN /hpf will falsely exclude 23.8% of patients with discharge and dysuria and 29% of patients with discharge alone, we include in our definition of NGU those patients with a urethral discharge and/or a count of more than four PMN /hpf in their urethral smear and a negative culture for N. gonorrhoeae. On this basis, 107 patients were entered into the NGU study group. The polygamous control group was defined to include men without urethral discharge and with fewer than four PMN /hpf and numbered 87 patients. Rates of isolation of microorganisms in patients with urethritis and in controls. Yeasts were isolated from only two of 100 patients studied for the presence of yeasts and viruses. C. albicans was isolated from one patient in the NGU study group, and Trichosporon from one of the polygamous control patients. H. hominis type 2 was isolated from two NGU study patients and one GU patient. CMV was not found in any
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Table 2. Isolation rates of anaerobes from patients with nongonococcal urethritis (NGU), patients with gonococcal urethritis (GU), and control patients. Controls Monogamous
Polygamous
Peptococcus Peptostreptococcus Propionibacterium-A rachnia Bacteroides Eubacterium Clostridium Streptococcus Velllonella Fusobacterium Lactobacillus
14/36 2/36 9/36 5/36 2/36 0 2/36 1/36 0 1/36
(2.8)
13/44 (29.5) 1/44 (2.3) 10/44 (22.7) 13/44 (29.5) 2/44 (4.6) 2/44 (4.6) 0 1/44 (2.3) 2/44 (4.6) 0
26/48 7/48 5/48 6/48 1/48 0 1/48 2/48 0 0
22/25 (88)
21/22 (95.5)
27/32 (84.4)
Overall isolation rate *
(38.9) (5.6) (25) (13.9) (5.6) (5.6) (2.8)
NGU study group (54.2) (14.6) (10.4) (12.5) (2.1) (2.1) (4.2)
GU group 14/32 (43.8) 6/32 (18.8) 1/32 (3.1) 8/32 (25) 0 2/32 (6.2) 1/32(3.1) 0 0 0 16/21 (76.2)
NOTE. Except where indicated otherwise, data are number of isolates of individual anaerobe/total number of isolates of anaerobes for group (percentage). *Data are number of patients from whom anaerobes were isolated/total number of patients in group (percentage).
and C. trachomatis-negative NGU study patients showed no evidence of dependence of group B streptococci or C. vaginale on the presence or absence of C. trachomatis (P = 0.530 and 0.622, respectively). Clinical factors influencing isolation rates. To determine whether the sequence in which the specimens were obtained by urethral swabbing had any relation to isolation rates, we calculated individual isolation rates per study group (NGU, GU, and clinic controls) per swab (first, second, and third). In all study groups the isolation rates for C. trachomatis, group B streptococci, and C. vaginale were always independent of the order in which the urethral swabs were obtained. The isolation rates of C. trachomatis, group B streptococci, and C. vaginale were not related to the time elapsed since last urination (P = 0.153, 0.541, and 0.161, respectively). Furthermore, the presence of these organisms was not related to
the number of different sexual partners III the past three months (P = 0.192, 0.913, and 0.422, respectively) or to the number of days since last sexual intercourse (P = 0.721, 0.363, and 0.259, respectively). Symptomatology in urethritis patients. Sixtysix percent of the NGU study patients had no demonstrable urethral discharge or minimal (1+) discharge. Of the NGU study patients with a urethral discharge, 65% exhibited clear or white discharge. In contrast, 81% of the GU patients had moderate to large (2+ or 3+) amounts of discharge, which was purulent in 84% of the cases. In the NGU study group, no dependence could be established between the amount or type of discharge and the presence of C. trachomatis in the urethral secretions (P = 0.091 and 0.551, respectively) (tables 4 and 5). In the GU group, as the amount of discharge increased, the rate of isolation of C. trachomatis decreased. In the NGU study group, there was no significant differ-
Table 3.
Rates of isolation of group B streptococci, Corynebacterium vaginale (Haemophilus vaginalis), and Chlamydia trachomatis from patients with nongonococcal urethritis (NGU), patients with gonococcal urethritis (GU), and control patients. Controls
Organism Group B streptococci C. vaginate C. trachoma tis
Monogamous 3/25(12) 2/25(8) 0/25
Polygamous 21/87(24.1 ) 13/60(21.7)* 6/87(6.9)
*Several specimens were omitted because of bacterial oyergrowth on agar media.
NGU study group 24/107(22.4) 19174(25.7)*
35/107 (32.7)
GU patients 11/61(18.0) 5/29( 17.2)* 12/61(19.6)
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Anaerobe
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Table 4. Amount of urethral discharge at the time of examination in patients with gonococcal urethritis (GD) and nongonococcal urethritis (NGD) correlated with detection of Chlamydia trachomatis.
Table 5. Type of urethral discharge at the time of examination in patients with nongonococcal urethritis (NGD) and gonococcal urethritis (GD) correlated with the incidence of detection of Chlamydia tracho-
matis. Urethral discharge Group
None
NGU GU
7{30 (23) 2{6 (33)
2+
3+
8{18 (44) 2{10(20)
5{17 (29) 5{37 (13.5)
1+
13{37 (35) 2{5 (40)
NOTE. Data are number of patients positive for C. trachomatis{number of patients with indicated amount of urethral discharge (percentage). See Materials and Methods for definition of scale for urethral discharge.
=
Group NGU GU
None
7{30 (23) 2{6 (33)
Clear
19{49 (39) 1{3 (33)
White
Yellow{ gray
5{17 (29) 2{5 (40)
2{6 (33) 6{44 (14)
NOTE. Data are number of patients positive for C. trachomatis{number of patients with indicated type of discharge (percentage).
trachomatis was unrelated to the number of PMN (P = 0.078); 90.9% of the 33 Chlamydiapositive patients had < 50 PMN jhpf, and 73.9% of the 69 Chlamydia-negative patients had < 50 PMN/hpf. The presence of dysuria for seven days or longer in the NCU study group correlated with the isolation of C. trachomatis (P = 0.025). In the CU group, no such relationship could be established (P = 0.117) (table 6). Discussion
Urethral discharge in male patients has been a generally accepted objective criterion in the diagnosis of urethritis. Our data indirectly support this criterion in that no urethral discharge was noted in any of our monogamous control patients. This study adds a second objective criterion in the diagnosis of urethritis, the observation of more than four PMN /hp£ in the urethral smear. Because the gram-stained smear of urethral secretions is a generally accepted proTable 6. Durations of urethral discharge and dysuria before the initial visit to the venereal disease clinic correlated with detection of Chlamydia trachomatis in patients with nongonococcal urethritis (NGD) and patients with gonococcal urethritis (GD). Urethral discharge
;;.7 days
Group
2 hr before examination, 75.9% had < 50 PMN jhpf. Likewise, no dependence could be demonstrated between the interval since last sexual intercourse and the number of PMN (P = 0.267); of the 58 NGU study patients who had had sexual contact within the past two days, 84.5% had < 50 PMN jhpf, and 72.7% of the 44 NGU study patients without sexual contact for two days had < 50 PMN jhpf. Isolation of C.
Type of urethral discharge
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not be described from our data. At present, our definition of NGU includes only those men with negative urethral cultures for N. gonorrhoeae who have a urethral discharge and/or more than four PMN /hpf in their urethral smears. Our results substantiate those of other investigators in showing an association of C. trachomatis with NGU [2, 31-34]. Excluding our 25 asymptomatic NGU patients from the total NGO study population so as to make our NGU group clinically comparable to that studied by Holmes et al. [2], we isolated C. trachomatis in 35.8% of our NGU patients, as compared with the rate of 42% found by Holmes et al. [2]. Transmission of C. vaginate and group B streptococci seems to relate to sexual activity, but the two organisms are not etiologically responsible for NGO, a situation comparable to that with M. hominis [2]. These organisms are found in comparable numbers in NGU, GU, and control patients matched for equal degrees of sexual activity, whereas in monogamous control patients they were found infrequently. Other sexually transmitted microorganisms, such as T. vaginate [2], are only infrequently isolated from patients with NGU. This study adds yeasts and confirms H. hominis [2] as sexually transmitted microorganisms not isolated from significant numbers of NGO patients. It is difficult to interpret the statistically significant higher isolation rates for the anaerobic organism Propionibacterium-Arachnia and the lower isolation rates for peptostreptococci in the combined monogamous-polygamous control groups vs. the combined NGU-GU groups, since overgrowth of normal aerobic urethral commensal organisms as a possible confounding factor was not determined. Since C. trachomatis can be cultured from 35%-40% of NGU patients, it seems reasonable to relate the presence of this organism to NGO symptomatology. Richmond et al. found that the isolation rate for C. trachomatis was significantly greater in men who had had urethral discharge for more than seven days [31]. Our results show no relation between isolation of C. trachoma tis and duration, amount, or type of discharge. Furthermore, we could not demonstrate a correlation between C. trachomatis isolation and the number of PMN /hpf on the urethral smear. These data suggest the possibility that some as
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cedure used in the laboratory diagnosis of urethritis [3], our PMN counts were determined from gram-stained smears of urethral secretions obtained by swabbing the urethra with calcium alginate swabs. This test, although semiquantitative relative to the accuracy achieved in measurements of PMN counts in peripheral blood by use of a hemocytometer, allows the clinician to measure PMN in the urethral smear and make a bacteriologic differential diagnosis of GU from NGU concurrently. By comparison of histograms of the PMN counts from NGU patients with dysuria and discharge and from polygamous men without dysuria or discharge (table 1), a count of four PMN /hpf was determined to be the cutoff point that differentiated patients with urethritis from those without urethritis with the highest degree of sensitivity and specificity. Furthermore, the PMN count was found to correlate with the amount of urethral discharge noted on physical examination, and the. PMN count was independent of the interval since last urination or the time since last sexual intercourse. This is the first attempt in the scientific literature to define systematically a PMN count that separates urethritis patients from nonurethritis patients. Other investigators [31, 32] have arbitrarily chosen a count of 10 PMN /hpf as the cutoff point without providing any justification for doing so. We feel that our method of analysis in arriving at four PMN /hpf as the separation point represents a significant improvement in the diagnosis ofNGo. We have found the use of four PMN /hpf most helpful in the evaluation of patients with complaints of dysuria but with no urethral discharge. Dysuria without demonstrable urethral discharge and with fewer than four PMN /hpf in the urethral smear occurred in 15.1% of our polygamous control population. C. trachomatis was isolated in 5.6% of these patients with dysuria alone, a rate significantly lower (P = 0.032) than the 32.3% isolation rate for C. trachomatis in patients with discharge and/or more than four PMN /hpf, but not significantly different from the 7% isolation rate in asymptomatic polygamous controls (P = 0.755). The pathophysiology of dysuria without the presence of urethral discharge and with fewer than four PMN /hpf can-
Swartz et al,
Nongonococcal Urethritis
yet unrecognized microorganism(s) may well be etiologic determinants of NGU, rather than that present culture techniques are inadequate to isolate C. trachomatis in the 60% of C. trachomatis-negative NGU patients. By using a cutoff point of four PMN /hpf in the definition of NGU, we have shown that asymptomatic NGU is more prevalent in a ve-
lated from 17% of our asymptomatic NGU patients as compared with 32.3«j'o of the total NGU study group. If follow-up evaluations of these patients show the C. trachomatis isolation rate approaching that for classic NGU, or if they develop symptomatology of urethritis, it is unlikely that the occurrence of urethral PMN is only a transient phenomenon of no clinical significance. In summary, a urethral discharge seen on physical examination has been the only objective criterion in the diagnosis of NGU. This finding was present in 78% of our NGlJ study patients. Our study demonstrates that a count of more than four PMN /hpf in the urethral smear can also be an objective criterion in the diagnosis of urethritis. The number of PMN /hpf is unaffected by the interval since last urination or the time since last intercourse. The finding of more than four PMN in the urethral smears of 22% of asymptomatic polygamous men suggests the not uncommon existence of asymptomatic NGU.
References 1. Volk, J., Kraus, S. J. Nongonococcal urethritis - a venereal disease as prevalent as epidemic gonorrhea. Arch. Intern. Med. 134:511-514,1974. 2. Holmes, K. K., Handsfield, H. H., Wang, S. P., Wentworth, B. B., Turck, M., Anderson, J. B., Alexander, E. R. Etiology of nongonococcal urethritis. N. Eng!. J. Med.292:I199-1205, 1975. 3. Jacobs, N. F., Jr., Kraus, S. J. Gonococcal and nongonococcal urethritis in men: clinical and laboratory differentiation. Ann. Intern. Med. 82:7-12, 1975.
4. Csonka, G. W. Non-gonococcal urethritis. Br. J. Vener. Dis. 41:1-8,1965. 5. Fowler, W. Studies in non-gonococcal urethritis therapy. The long-term value of tetracycline. Br. J. Vener. Dis. 46:464-468,1970. 6. Boyd, J. T., Csonka, G. W., Oates, J. K. Epidemiology of non-specific urethritis. Br. J. Vener. Dis. 34:40-43, 1958. 7. Arum, E. S., Jacobs, N. F., Jr. Nongonococcal urethritis. Cutis 17:719-722, 1976. 8. Swartz, S. L. Diagnosis of nongonococcal urethritis. In K. K. Holmes and D. Hobson [ed.], Nongonococcal urethritis and related oculogenital infections. American Society of Microbiology, Washington, D.C., 1977, p. 15-18. 9. Center for Disease Contro!. Recommended treatment schedules for gonorrhea. Ann. Intern. Med. 76:991, 1972. 10. Dunkelberg, W. E., Jr., McVeigh, I. Growth requirements of Haemophilus vaginal is. Antonie van Leeuwenhoek 35:129-145,1969. II. Dunkelberg, W. E., Jr., Skaggs, R., Kellogg, D. S., Jr. A study and new description of Corynebacterium vaginale (Haemophilus vaginalis). Am. J. CHn. Patho!' 53:370-377,1970. 12. Dukes, C. D., Gardner, H. L. Identification of Haemophilus vaginalis. J. Bacterio!. 81:277-283, 1961. 13. Malone, B. H., Schreiber, M., Schneider, N. J., Holdeman, L. V. ObHgately anaerobic strains of Corynebacterium vagina le (Haemophilus vaginalis). J. CHn. Microbio!. 2:272-275, 1975. 14. Facklam, R. R., Padula, J. F., Thacker, L. G., Wortham, E. C., Sconyers, B. J. Presumptive identification of group A, B, and D streptococci. App!. Microbio!. 27:107-113,1974. 15. Swift, H. F., Wilson, A. T., Lancefield, R. C. Typing group A hemolytic streptococci by M precipitin reactions in capillary pipettes. J. Exp. Med. 78:127-133, 1943. 16. Wilkinson, H. W., Facklam, R. R., Wortham, E. C. Distribution by serological type of group B streptococci isolated from a variety of clinical material over a five-year period (with special reference to neonatal sepsis and meningitis). Infec. Immun. 8:228-235, 1973. 17. Jacobs, N. F., Jr., Kraus, S. J. Comparison of hemoglobin-free culture media and Thayer-Martin medium for the primary isolation of Neisseria gonorrhoeae. J. Clin. Microbio!. 1:401-404,1975. 18. Kovalchik, M. T., Kraus, S. J. Neisseria gonorrhoeae: colonial morphology of rectal isolates. App!. Microbioi. 23:986-989, 1972. 19. Brown, W. J. Modification of the rapid fermentation test for Neisseria gonorrhoeae. App!. Microbio!. 27: 1027-1030, 1974. 20. Wentworth, B. B., Alexander, E. R. Isolation of Chlamydia trachoma tis by use of 5-iodo-2-deoxyuridinetreated cells. App!. Microbio!. 27:912-916,1974. 21. Reeve, P., Owen, J., Oriel, J. D. Laboratory procedures for the isolation of Chlamydia trachoma tis from the
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nereal disease clinic population than was previously thought [35]. At present, the clinical status of these patients is unclear in terms of determining the need for treatment for them and their sexual partners. The occurrence of urethral PMN in asymptomatic men may only be a transient phenomenon of no clinical significance, or it may be an expression of either an incubating or an asymptomatic phase of NGU (analogous to asymptomatic GU). C. trachomatis was iso-
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22.
23.
24.
25.
27.
28.
p.
29. Ostle, B. Statistics in research: basic concepts and techniques for research workers. 2nd ed. The Iowa State University Press, Ames, Iowa, 1963.585 p. 30. Snedecor, G. W., Cochran, W. G. Statistical methods. 6th ed. The Iowa State University Press, Ames, Iowa, 1967.593 p. 31. Richmond, S. J., Hilton, A. L., Clarke, S. K. R. Chlamydial infection: role of Chlamydia subgroup A in nongonococcal and post-gonococcal urethritis. Br. J. Vener. Dis. 48:437-444,1972. 32. Oriel, J. D., Reeve, P., Wright, J. T., Owen, J. Chlamydial infection of the male urethra. Br. J. Vener. Dis. 52:46-51, 1976. 33. Oriel, J. D., Reeve, P., Powis, P., Miller, A., Nicol, C. S. ChlamydiaI infection: isolation of Chlamydia from patients with non-specific genital infection. Br, J. Vener. Dis. 48:429-436, 1972. 34. Dunlop, E. M. C., Hare, M. J., Darougar, S., Jones, B. R. ChlamydiaI infections of the urethra in men presenting because of "non-specific" urethritis. In R. L. Nichols [ed.], Trachoma and related disorders caused by chlamydial agents. International Congress Series no. 233. Excerpta Medica, Amsterdam, 1971, p. 494500. 35. Roden, P. Asymptomatic non-specific urethritis. Br. J. Vener. Dis. 47:452-453, 1971.
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26.
human genital tract. J. Clin. Patho!' 28:910-914, 1975. Ahearn, D. G. Identification and ecology of yeasts of medical importance. In J. E. Prier and H. Friedman [ed.]. Opportunistic pathogens. University Park Press, Baltimore, 1974, p. 129-146. Bowman, P. I., Ahearn, D. G. Evaluation of the UniYeast-Tek kit for the identification of medically important yeasts. J. Clin. Microbio!. 2:354-358, 1975. Tokurnaru, T. Herpesvirus hominis. In E. H. Lennette and N. J. Schmidt [ed.], Diagnostic procedures for viral and rickettsial infections. 4th ed. American Public Health Association, New York, 1969, p. 641676. Bernstein, M. T., Stewart, J. A. Method for typing antisera to Herpesvirus hominis by indirect hemagglutination inhibition. App!. Microbio!' 21:680-684,1971. Dowell, V. R. Jr. Methods for isolation of anaerobes in the clinical laboratory. Am. J. Med. Techno!. 41:402410,1975. Martin, W. J. Practical methods for isolation of anaerobic bacteria in the clinical laboratory. App!. Microbioi. 22:1168-1171, 1971. Dowell, V. R., j-, Hawkins, T. M. Laboratory methods in anaerobic bacteriology. Department of Health, Education, and Welfare publication no. (CDC) 748272. Center for Disease Control, Atlanta, 1974. 96
Diagnosis and Etiology of Nongonococcal Urethritis Stephen L. Swartz, Stephen J. Kraus, Kenneth L. Herrmann, Michael D. Stargel, W. Jerry Brown, and Stephen D. Allen
From the Venereal Disease Research Branch and the Enterobacteriology Branch, Bacteriology Division, and the Perinatal Virology Branch, Virology Division, Center fOT Disease Control, Atlanta, Georgia
Nongonococcal urethritis (NGU) presently accounts for more than half of the cases of urethritis seen in sexually active males attending venereal disease clinics in the United States [1, 2]. Diagnosis of NGU is currently one of exclusion [3], the therapy is controversial [4, 5], and the incubation period [6] and management of sexual partners are uncertain [7]. Clinical studies and management of patients with NGU would be aided if NGU were more clearly defined and its etiology were better understood [8]. Inadequate criteria are available to differentiate patients with minimal symptomatology of urethritis from patients with "psychosomatic urethritis" who have no objective evidence of urethral inflammation on
physical and laboratory examination. One objective of this study was to determine the minimal number of polymorphonuclear cells (PMN) that must be present in a gram-stained smear of urethral secretion to distinguish physiologic urethral exudate from pathologic urethral discharge. Previous attempts to determine the etiology of NGU have focused on Chlamydia trachomaiis,
Mycoplasma hominis, Ureaplasma urealsticum, Herpesvirus hominis, and Trichomonas vaginale. At present, only C. trachomatis and possibly U. urealyticum have been implicated as potential causes of NGU in appropriately controlled studies. The second objective of this study was to examine the occurrence of other microorganisms in patients with NGU.
Received for publication June 20, 1977. and in revised form April 13, 1978. We thank Dr. Donald G. Ahearn, Georgia State University, for yeast isolation and identification; Jay S. Smith, Statistical Activities Branch, Center for Disease Control, for statistical assistance; and Pamela Routh, Lillian Cooper, and Rex Poole of the DeKalb County Venereal Disease Clinicfor their assistance. Useof trade names is for identification only and does not constitute endorsement by the U.S. Public Health Service or by the U.S. Department of Health, Education,and Welfare. Please address requests for reprints to the Center for Disease Control. Technical Information Services, Bureau of StateServices, Atlanta, Georgia 30333.
Materials and Methods
Study population. The study population included 280 men attending the DeKalb County Venereal Disease Clinic, Atlanta, Ga., from October 1975 through March 1976. Clinic patients were excluded if they had received any antibiotics within the preceding four weeks, if they had a visible deformity of the urethral meatus on physical examination, or if they failed to give in-
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The observation of more than four polymorphonuclear cells (PMN) per high-power field (hpf) in gram-stained smears of urethral secretions was found to differentiate patients with urethritis from patients without urethritis. A urethral discharge was present in 78% of patients with nongonococcal urethritis (NGU). Dysuria without demonstrable urethral discharge and with fewer than four PMN /hpf did not appear to fit into the NGU spectrum. NGU is now defined to include men who have negative urethral cultures for Neisseria gonorrhoeae with a urethral discharge and/or more than four PMN /hpf in their urethral smears. The finding of more than four PMN /hpf in the urethral smears of 22% of asymptomatic sexually active men with more than one sexual partner (polygamous controls) suggeststhat asymptomatic NGU is not uncommon. Chlamydia trachoma tis was isolated significantly more frequently from the NGU study group than from the control group (P < 0.001). This study adds Corynebacterium vaginale (Haemophilus vaginalis) , group B streptococci, and yeasts to the list of sexually transmitted microorganisms that are not etiologic determinants of NGU.
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=
Hewitt broth for subsequent culture of group B streptococci. A second swab was inserted I em further into the anterior urethra, inoculated directly onto VCN medium (Thayer-Martin media without hemoglobin) for culture of Neisseria gonorrhoeae, and then placed in sucrose-phosphate transport media for C. trachomatis. A third swab was obtained in the same manner, inoculated directly onto Sabouraud's media for yeasts, and then placed in I ml of Eagle's minimal essential medium (MEM) for viral studies. After reviewing the results from the initial 100 samples, we decided that the incidence of fungal and viral isolation was too low to justify analyzing the third swab for such organisms, and for the rest of the study, these swabs were directly inoculated onto prereduced anaerobic media and immediately placed in an oxygen-free environment. Each 2- X l-cm smear of urethral secretion was gram-stained and examined for the presence of PMN and gram-negative diplococci. The slide was first scanned under low power (x 100) to locate areas with the highest concentration of PMN. These areas were then examined under high magnification (x970), and the number of PMN in the five microscopic fields with the highest number of PMN were counted and averaged. Preliminary studies have shown that while the total number of PMN in many areas on a slide averaging fewer than four PMN jhpf may outnumber the total number of PMN on a slide averaging four or more PMN jhpf, only rarely «1.5% of slides examined) does this situation occur. Consequently, this finding need not be considered a problem in our laboratory evaluation of urethral specimens. Identification of microorganisms by culture. C. vaginale. The PSD medium [10, II] was modified by the addition of I ml of IsoVitalex" (Baltimore Biological Laboratories [BBL], Cockeysville, Md.)jI00 ml of medium, an addition which facilitated growth of certain isolates of C. vaginale that grew poorly on standard PSD media. After the plates were inoculated with the urethral sample, they were incubated at 36.5 C for 48 hr in a candle-extinction jar. C. vaginale was identified on the basis of (1) colonial morphology as seen at a magnification of x40 with a stereoscopic microscope [II]; (2) inhibition of
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formed written consent. All other male patients were prospective candidates since this study included men with gonococcal urethritis (GU), men with NGU, and men without urethritis who were sexually active and had more than one sexual partner (polygamous). The last group included men who were seen at the clinic either for routine examination or for evaluation of external genital lesions such as condylomata acuminata, scabies, pediculosis pubis, or syphilis. The study also included a monogamous control group consisting of 25 male volunteers who were matched by age to the clinic population. Each of these volunteers (1) had had sexual intercourse with only one person during the preceding 90 days, (2) was free of symptoms of urethritis, and (3) had no urethral discharge on physical examination. Patients with gonorrhea received either aqueous procaine pencillin G plus probenecid or tetracycline hydrochloride according to the recommendations of the U.S. Public Health Service [9], and men with NGU were treated with 500 mg of tetracycline hydrochloride four times a day for seven days. Evaluation and collection of urethral specimens. All patients were interviewed and examined by the same physician (S.L.S.). Any urethral discharge was noted and quantitated as follows: 0 = no discharge present after penile stripping (i.e., compressing the urethra from the bulb distally); 1+ = no spontaneous discharge, but slight discharge present after penile stripping; 2+ = no spontaneous discharge, but moderate discharge present after penile stripping; 3+ spontaneous discharge present. Three urethral specimens were obtained in random sequence by insertion of calcium alginate swabs into the urethra. A preliminary study of NGU had shown that if each swab was inserted -I em further into the urethra, the numbers of PMN per high-power field (hpf) from three consecutive swabs were not significantly different. A gram stain was prepared from each swab by rolling the swab once over a 2- X l-cm area on a glass slide to achieve as uniform a dispersion of PMN over the slide as possible; the swab was then inoculated onto a plate of modified PSD media (protease peptone, starch, and dextrose) for culture of Corynebacterium vaginale (Haemophilus vaginalis) and placed in 5 ml of Todd-
Nongonococcal Urethritis
blast cells. The culture tubes were transported to the laboratory within 6 hr of inoculation and incubated at 37 C [24]. The cultures were observed daily for one week for evidence of CPE characteristic of H. hominis infection and then weekly for one month for evidence of CPE characteristic of CMV infection. H. hominis isolates were typed with use of indirect HAl [25]. A naerobes. All urethral swabs to be analyzed for anaerobic organisms were inoculated immediately in the clinic onto one plate of blood agar (5% rabbit blood in trypticase soy agar containing yeast extract, vitamin K1, and hemin) [26], one plate of the same medium modified by the addition of neomycin (final concentration, 100 g/ml), and a tube of thioglycolate broth (BBL 0135C) supplemented with 10 g of heminjrnl and 10 g of vitamin K1/ml. The media were prereduced for at least 18 hr in an atmosphere of 85% N 2 , 10% H 2 , and 5% CO 2 before use. Exposure to oxygen during inoculation was minimized by a holding-jar procedure modified from that of Martin [27] by use of a mixture of 85% N 2 , 10% H 2 , and 5% CO 2 passed through a palladium catalyst and by use of the holding jars at the time of primary inoculation in the clinic. Following inoculation, all plates and tubes were incubated for five days at 35 C in GasPak jars with disposable GasPak H 2-C0 2 generators (BBL). After incubation each plate was carefully examined under a dissecting microscope, and with the aid of a modified holding-jar technique, one or more representatives of each colony type were picked (usually four to eight colonies per plate) for anaerobic subculture and purification on blood agar. The pure-culture isolates were characterized by colonial and gram-stain morphology, presence or absence of endospores, aerotolerance on blood agar plates, and gas-liquid chromatographic analysis of metabolic products in Schaedler broth (BBL) culture (4872 hr) using the procedures of Dowell and Hawkins [28]. The identification of genera reported here was only presumptive and was based on the data of Dowell and Hawkins [28]. A X2 test was applied to all contingency tables [29] to check for the degree of independence among the variables of interest. The level of significance was chosen to be 5% in all cases. Yates's
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growth by 3.0% H 202 [11]; (3) a negative catalase reaction [11]; (4) a gram stain showing groups of variably staining small coccoids and short pleomorphic rods [12]; (5) incomplete phemolysis of freshly prepared rabbit blood agar [13]; and (6) fermentation of dextrose, maltose, and starch, but no fermentation of mannitol or rhamnose [13]. All C. vaginale isolates were confirmed by Dr. W. E. Dunkelberg, Medical Laboratory Detachment, Dwight David Eisenhower Army Medical Center, Fort McPherson, Ga. Group B streptococci. From the test tube containing 5 ml of Todd-Hewitt broth, pour and streak plates were made for isolation of pure colonies of group B streptococci [I4]. Pure cultures of these isolated colonies were extracted by the hot HCl method of Swift et al. [I5]. The extracted streptococcal antigen was added to specific grouping and typing antisera in sterile capillary tubes, and the appearance of a precipitin reaction was noted [16]. N. gonorrhoeae. Gonococci in specimens inoculated onto VCN medium [I7] were identified by standard techniques [I8, 19]. C. trachomatis. The sucrose-phosphate transport tubes were kept frozen at -70 C until the time of inoculation. McCoy cells, treated with 5iodo-2'-deoxyuridine according to the technique of Wentworth and Alexander [20], were grown as monolayers on cover slips in I-dram vials. The clinical specimens were inoculated onto these cover slips, centrifuged at 3,000 g at 35 C for 60 min [21], and incubated for three days. The cover slips were fixed in methanol, stained with Jones-iodine solution [20], and examined at X 400 magnification for typical intracytoplasmic inclusions. Yeasts. Mycological agar (Difco, Detroit, Mich.) fortified with 0.5 g of chloramphenicol was used for the primary isolation of yeasts. All isolates were screened by the germ-tube test for the presence of Candida albicans [22]. A germ tube-negative isolate and a germ tube-positive isolate were speciated with the Uni-Yeast-Tek system (Corning Medical, Corning, N.Y.) [23]. Viruses. Specimens for isolation of H. hominis and cytomegalovirus (CMV) were placed immediately onto I ml of Eagle's MEM for 15-30 min and then inoculated directly onto rollertube cultures of human embryonic lung fibro-
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correction was used where cell sizes were fewer than five patients in two-way tables [30]. Fisher's exact probability is not given because of the number of observations available. Results
Numbers of PMN in urethral specimens. The mean number of PMN jhpf was determined for each of 299 slides. Of the 61 patients with a diagnosis of gonorrhea (gonorrhea being defined as urethritis from which N. gonorrhoeae was cultured), all but one had ee 50 PMN jhpf and typical gram-negative diplococci usually located intracellularly but occasionally only extracellularly. The 25 monogamous controls were without discharge or dysuria and had an average count (±SE) of 0.8 ± 0.7 PMN jhpf (range, 0-2.2). A histogram describing the PMN counts in urethral smears for polygamous men without discharge or dysuria and for NGU patients (with both discharge and dysuria) is presented in figure 1. Although some NGU patients had large 80
1
78:1 26 24
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16
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Polygamous Men without Dysuria or Discharge
NGU with Dysuria and Discharge
:;
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*
14 12 10 8 6 4
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8
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40
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48
52
56
60
64
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68
72
76
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84
88
92
96
100
No. of PMN/hpf
Figure 1. Histogram of counts of polymorphonuclear cells (PMN) per high-power field (hpf) in urethral smears from patients with nongonococcal urethritis (NGU) with dysuria and discharge and from men without dysuria or discharge who were sexually active and had more than one sexual partner (polygamous control group).
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numbers of urethral PMN, an overlap in the number of PMN /hpf existed among many other NGU patients and the polygamous men without signs or symptoms of urethritis. For determination of the number of PMN jhpf that would best separate patients into these two groups, a table was constructed in which the counts of PMN jhpf for both groups are given in consecutive integers as cutoff points (table 1). To obtain the highest degree of sensitivity and specificity in choosing a PMN count that separates patients with urethritis from those without urethritis, we selected the row showing the minimal percentages of false-negative results and false-positive results (i.e., where the difference between these values was the smallest). This row corresponded to a count of four PMN jhpf. When a histogram for the 31 patients with urethral discharge alone was constructed, 29% had fewer than four PMN. This false-negative rate is similar to that of 23.8% obtained for "typical" NGU patients with both discharge and dysuria. However, when the histogram for the 26 patients
449
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of the patients. Among the 100 patients studied for the presence of anaerobic organisms, no significant differences in overall rates of isolation were noted for the GU group, the NGU study group, and the polygamous and monogamous control groups (P = 0.575) (table 2). An average of seven different anaerobic genera were isolated from each of these groups. Comparison of isolation rates for individual anaerobic organisms revealed statistically higher isolation rates for Propionibacterium-Arachnia (P < 0.001) and lower isolation rates for peptostreptococci (P = 0.024) in the combined monogamous and polygamous control groups than in the combined NGU and GU groups. Group B streptococci and C. vaginale were isolated from the NGU, GU, and polygamous control patients at statistically comparable rates (P = 0.670 and 0.705, respectively) (table 3). For the group B streptococci, the isolation rates for all serotypes were similar within each of the patient populations, with serotype III always the most prevalent. C. trachomatis was isolated significantly more frequently from the NGU study group than from the polygamous control group (P < 0.001) (table3). Analysis of the isolation rates of group B streptococci and C. vaginale in C. trachomatis-positive
Table 1. Counts of polymorphonuclear cells (PMN) in urethral smears from patients with nongonococcal urethritis (NGU) having dysuria and urethral discharge and from men without dysuria or discharge who were sexually active and had more than one sexual partner (polygamous control group).
Cutoff count ofPMN 1
2 3 4 5 6 7 8 9 10 11 12 13
14 15 16
NGo patients (percentage of false-negative results) 15.2 17.4 21.7 23.8 28.1 30.3 32.3 32.3 34.4 43.1 47.4 47.4
49.5 53.8 53.8 53.8
Percentage of false-positive results (patients with asymptomatic NGU)* 37.2 27.7 23.8 22.0 19.3 18.2 17.2 16.3 12.5 10.6 8.6 7.8 5.8 5.0 4.0 1.2
*Calculated by 100% minus the value for polygamous men without dysuria or discharge.
Polygamous men without dysuria or discharge (cumulative percentages) 62.8 72.3 76.2 78.0 80.7 81.8 82.8 83.7 87.5 89.4 91.4
92.2 94.2 95.0 %.6 93.8
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with dysuria alone was analyzed, 76.9% had fewer than four PMN. This value is similar to the 78% of polygamous men without discharge or dysuria who had fewer than four PMN and suggests that dysuria alone cannot be used to place patients in the NGU group. Recognizing that a definition of NGU based solely on a count of more than four PMN /hpf will falsely exclude 23.8% of patients with discharge and dysuria and 29% of patients with discharge alone, we include in our definition of NGU those patients with a urethral discharge and/or a count of more than four PMN /hpf in their urethral smear and a negative culture for N. gonorrhoeae. On this basis, 107 patients were entered into the NGU study group. The polygamous control group was defined to include men without urethral discharge and with fewer than four PMN /hpf and numbered 87 patients. Rates of isolation of microorganisms in patients with urethritis and in controls. Yeasts were isolated from only two of 100 patients studied for the presence of yeasts and viruses. C. albicans was isolated from one patient in the NGU study group, and Trichosporon from one of the polygamous control patients. H. hominis type 2 was isolated from two NGU study patients and one GU patient. CMV was not found in any
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Table 2. Isolation rates of anaerobes from patients with nongonococcal urethritis (NGU), patients with gonococcal urethritis (GU), and control patients. Controls Monogamous
Polygamous
Peptococcus Peptostreptococcus Propionibacterium-A rachnia Bacteroides Eubacterium Clostridium Streptococcus Velllonella Fusobacterium Lactobacillus
14/36 2/36 9/36 5/36 2/36 0 2/36 1/36 0 1/36
(2.8)
13/44 (29.5) 1/44 (2.3) 10/44 (22.7) 13/44 (29.5) 2/44 (4.6) 2/44 (4.6) 0 1/44 (2.3) 2/44 (4.6) 0
26/48 7/48 5/48 6/48 1/48 0 1/48 2/48 0 0
22/25 (88)
21/22 (95.5)
27/32 (84.4)
Overall isolation rate *
(38.9) (5.6) (25) (13.9) (5.6) (5.6) (2.8)
NGU study group (54.2) (14.6) (10.4) (12.5) (2.1) (2.1) (4.2)
GU group 14/32 (43.8) 6/32 (18.8) 1/32 (3.1) 8/32 (25) 0 2/32 (6.2) 1/32(3.1) 0 0 0 16/21 (76.2)
NOTE. Except where indicated otherwise, data are number of isolates of individual anaerobe/total number of isolates of anaerobes for group (percentage). *Data are number of patients from whom anaerobes were isolated/total number of patients in group (percentage).
and C. trachomatis-negative NGU study patients showed no evidence of dependence of group B streptococci or C. vaginale on the presence or absence of C. trachomatis (P = 0.530 and 0.622, respectively). Clinical factors influencing isolation rates. To determine whether the sequence in which the specimens were obtained by urethral swabbing had any relation to isolation rates, we calculated individual isolation rates per study group (NGU, GU, and clinic controls) per swab (first, second, and third). In all study groups the isolation rates for C. trachomatis, group B streptococci, and C. vaginale were always independent of the order in which the urethral swabs were obtained. The isolation rates of C. trachomatis, group B streptococci, and C. vaginale were not related to the time elapsed since last urination (P = 0.153, 0.541, and 0.161, respectively). Furthermore, the presence of these organisms was not related to
the number of different sexual partners III the past three months (P = 0.192, 0.913, and 0.422, respectively) or to the number of days since last sexual intercourse (P = 0.721, 0.363, and 0.259, respectively). Symptomatology in urethritis patients. Sixtysix percent of the NGU study patients had no demonstrable urethral discharge or minimal (1+) discharge. Of the NGU study patients with a urethral discharge, 65% exhibited clear or white discharge. In contrast, 81% of the GU patients had moderate to large (2+ or 3+) amounts of discharge, which was purulent in 84% of the cases. In the NGU study group, no dependence could be established between the amount or type of discharge and the presence of C. trachomatis in the urethral secretions (P = 0.091 and 0.551, respectively) (tables 4 and 5). In the GU group, as the amount of discharge increased, the rate of isolation of C. trachomatis decreased. In the NGU study group, there was no significant differ-
Table 3.
Rates of isolation of group B streptococci, Corynebacterium vaginale (Haemophilus vaginalis), and Chlamydia trachomatis from patients with nongonococcal urethritis (NGU), patients with gonococcal urethritis (GU), and control patients. Controls
Organism Group B streptococci C. vaginate C. trachoma tis
Monogamous 3/25(12) 2/25(8) 0/25
Polygamous 21/87(24.1 ) 13/60(21.7)* 6/87(6.9)
*Several specimens were omitted because of bacterial oyergrowth on agar media.
NGU study group 24/107(22.4) 19174(25.7)*
35/107 (32.7)
GU patients 11/61(18.0) 5/29( 17.2)* 12/61(19.6)
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Anaerobe
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Nongonococcal Urethritis
Table 4. Amount of urethral discharge at the time of examination in patients with gonococcal urethritis (GD) and nongonococcal urethritis (NGD) correlated with detection of Chlamydia trachomatis.
Table 5. Type of urethral discharge at the time of examination in patients with nongonococcal urethritis (NGD) and gonococcal urethritis (GD) correlated with the incidence of detection of Chlamydia tracho-
matis. Urethral discharge Group
None
NGU GU
7{30 (23) 2{6 (33)
2+
3+
8{18 (44) 2{10(20)
5{17 (29) 5{37 (13.5)
1+
13{37 (35) 2{5 (40)
NOTE. Data are number of patients positive for C. trachomatis{number of patients with indicated amount of urethral discharge (percentage). See Materials and Methods for definition of scale for urethral discharge.
=
Group NGU GU
None
7{30 (23) 2{6 (33)
Clear
19{49 (39) 1{3 (33)
White
Yellow{ gray
5{17 (29) 2{5 (40)
2{6 (33) 6{44 (14)
NOTE. Data are number of patients positive for C. trachomatis{number of patients with indicated type of discharge (percentage).
trachomatis was unrelated to the number of PMN (P = 0.078); 90.9% of the 33 Chlamydiapositive patients had < 50 PMN jhpf, and 73.9% of the 69 Chlamydia-negative patients had < 50 PMN/hpf. The presence of dysuria for seven days or longer in the NCU study group correlated with the isolation of C. trachomatis (P = 0.025). In the CU group, no such relationship could be established (P = 0.117) (table 6). Discussion
Urethral discharge in male patients has been a generally accepted objective criterion in the diagnosis of urethritis. Our data indirectly support this criterion in that no urethral discharge was noted in any of our monogamous control patients. This study adds a second objective criterion in the diagnosis of urethritis, the observation of more than four PMN /hp£ in the urethral smear. Because the gram-stained smear of urethral secretions is a generally accepted proTable 6. Durations of urethral discharge and dysuria before the initial visit to the venereal disease clinic correlated with detection of Chlamydia trachomatis in patients with nongonococcal urethritis (NGD) and patients with gonococcal urethritis (GD). Urethral discharge
;;.7 days
Group
2 hr before examination, 75.9% had < 50 PMN jhpf. Likewise, no dependence could be demonstrated between the interval since last sexual intercourse and the number of PMN (P = 0.267); of the 58 NGU study patients who had had sexual contact within the past two days, 84.5% had < 50 PMN jhpf, and 72.7% of the 44 NGU study patients without sexual contact for two days had < 50 PMN jhpf. Isolation of C.
Type of urethral discharge
452
not be described from our data. At present, our definition of NGU includes only those men with negative urethral cultures for N. gonorrhoeae who have a urethral discharge and/or more than four PMN /hpf in their urethral smears. Our results substantiate those of other investigators in showing an association of C. trachomatis with NGU [2, 31-34]. Excluding our 25 asymptomatic NGU patients from the total NGO study population so as to make our NGU group clinically comparable to that studied by Holmes et al. [2], we isolated C. trachomatis in 35.8% of our NGU patients, as compared with the rate of 42% found by Holmes et al. [2]. Transmission of C. vaginate and group B streptococci seems to relate to sexual activity, but the two organisms are not etiologically responsible for NGO, a situation comparable to that with M. hominis [2]. These organisms are found in comparable numbers in NGU, GU, and control patients matched for equal degrees of sexual activity, whereas in monogamous control patients they were found infrequently. Other sexually transmitted microorganisms, such as T. vaginate [2], are only infrequently isolated from patients with NGU. This study adds yeasts and confirms H. hominis [2] as sexually transmitted microorganisms not isolated from significant numbers of NGO patients. It is difficult to interpret the statistically significant higher isolation rates for the anaerobic organism Propionibacterium-Arachnia and the lower isolation rates for peptostreptococci in the combined monogamous-polygamous control groups vs. the combined NGU-GU groups, since overgrowth of normal aerobic urethral commensal organisms as a possible confounding factor was not determined. Since C. trachomatis can be cultured from 35%-40% of NGU patients, it seems reasonable to relate the presence of this organism to NGO symptomatology. Richmond et al. found that the isolation rate for C. trachomatis was significantly greater in men who had had urethral discharge for more than seven days [31]. Our results show no relation between isolation of C. trachoma tis and duration, amount, or type of discharge. Furthermore, we could not demonstrate a correlation between C. trachomatis isolation and the number of PMN /hpf on the urethral smear. These data suggest the possibility that some as
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cedure used in the laboratory diagnosis of urethritis [3], our PMN counts were determined from gram-stained smears of urethral secretions obtained by swabbing the urethra with calcium alginate swabs. This test, although semiquantitative relative to the accuracy achieved in measurements of PMN counts in peripheral blood by use of a hemocytometer, allows the clinician to measure PMN in the urethral smear and make a bacteriologic differential diagnosis of GU from NGU concurrently. By comparison of histograms of the PMN counts from NGU patients with dysuria and discharge and from polygamous men without dysuria or discharge (table 1), a count of four PMN /hpf was determined to be the cutoff point that differentiated patients with urethritis from those without urethritis with the highest degree of sensitivity and specificity. Furthermore, the PMN count was found to correlate with the amount of urethral discharge noted on physical examination, and the. PMN count was independent of the interval since last urination or the time since last sexual intercourse. This is the first attempt in the scientific literature to define systematically a PMN count that separates urethritis patients from nonurethritis patients. Other investigators [31, 32] have arbitrarily chosen a count of 10 PMN /hpf as the cutoff point without providing any justification for doing so. We feel that our method of analysis in arriving at four PMN /hpf as the separation point represents a significant improvement in the diagnosis ofNGo. We have found the use of four PMN /hpf most helpful in the evaluation of patients with complaints of dysuria but with no urethral discharge. Dysuria without demonstrable urethral discharge and with fewer than four PMN /hpf in the urethral smear occurred in 15.1% of our polygamous control population. C. trachomatis was isolated in 5.6% of these patients with dysuria alone, a rate significantly lower (P = 0.032) than the 32.3% isolation rate for C. trachomatis in patients with discharge and/or more than four PMN /hpf, but not significantly different from the 7% isolation rate in asymptomatic polygamous controls (P = 0.755). The pathophysiology of dysuria without the presence of urethral discharge and with fewer than four PMN /hpf can-
Swartz et al,
Nongonococcal Urethritis
yet unrecognized microorganism(s) may well be etiologic determinants of NGU, rather than that present culture techniques are inadequate to isolate C. trachomatis in the 60% of C. trachomatis-negative NGU patients. By using a cutoff point of four PMN /hpf in the definition of NGU, we have shown that asymptomatic NGU is more prevalent in a ve-
lated from 17% of our asymptomatic NGU patients as compared with 32.3«j'o of the total NGU study group. If follow-up evaluations of these patients show the C. trachomatis isolation rate approaching that for classic NGU, or if they develop symptomatology of urethritis, it is unlikely that the occurrence of urethral PMN is only a transient phenomenon of no clinical significance. In summary, a urethral discharge seen on physical examination has been the only objective criterion in the diagnosis of NGU. This finding was present in 78% of our NGlJ study patients. Our study demonstrates that a count of more than four PMN /hpf in the urethral smear can also be an objective criterion in the diagnosis of urethritis. The number of PMN /hpf is unaffected by the interval since last urination or the time since last intercourse. The finding of more than four PMN in the urethral smears of 22% of asymptomatic polygamous men suggests the not uncommon existence of asymptomatic NGU.
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nereal disease clinic population than was previously thought [35]. At present, the clinical status of these patients is unclear in terms of determining the need for treatment for them and their sexual partners. The occurrence of urethral PMN in asymptomatic men may only be a transient phenomenon of no clinical significance, or it may be an expression of either an incubating or an asymptomatic phase of NGU (analogous to asymptomatic GU). C. trachomatis was iso-
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