CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHOEAE are the primary bacterial pathogens causing urethral infection in men younger than age 35, and if not treated, place female partners at risk for these sexually transmitted diseases (STDs) and their associated upper genital tract complications. In addition, some men may progress to epididymitis. Use of highly sensitive and specific nucleic acid amplification testing (NAAT) for these organisms has led to increased appreciation of the frequent asymptomatic nature of these infections.1 Currently, therapy for male urethral infections principally relies on documentation of urethral symptoms, discharge, or evidence of inflammation on Gram stain of urethral swab specimens (≥5 polymorphonuclear leukocytes [PMNs] per oil-immersion field [oif]) or in first void urine (positive leukocyte esterase test).2 Without evidence of urethral inflammation or clinical findings of urethritis, empirical therapy is routinely deferred in asymptomatic men, as is testing in some clinical settings.
Until simple, highly sensitive and specific point-of-care assays are available for chlamydia and gonorrhea diagnosis, urethral Gram stain will remain a widely used tool for urethral infection screening and to guide decisions for empirical therapy. Earlier studies assessing Gram stain as a screening tool for urethral infection were usually based on chlamydia or gonorrhea diagnosis by culture. NAAT adds increased sensitivity over such prior tests, particularly in asymptomatic individuals who may have a low burden of organisms.3,4 Such assays provide an opportunity to better understand the relationship of urethral PMNs to chlamydia and gonorrhea.
We used an STD clinic database to analyze the relationship of urethral Gram stain findings to clinical characteristics and urethral infections and sought to determine how such relationships differed when diagnosis was based on NAAT versus culture. Further, we discuss the implications of our findings on the diagnosis and management of urethral chlamydial and gonococcal infections.
Materials and Methods
The study population was composed of men attending the Jefferson County Department of Public Health (JCDH) STD Clinic from February 1998 to August 2002 for routine evaluation (STD screening or symptom evaluation) who had both a urethral Gram stain and chlamydial and gonococcal NAAT performed. A subpopulation of men for who both culture and NAAT were performed was also evaluated; this group had culture performed as part of routine testing, yet had an extra specimen collected to test a NAAT for research purposes (the subpopulation did not differ from the overall study population in terms of reason for evaluation as all men presented for routine evaluation and were studied irrespective of presence of symptoms, signs, or known contact to STDs). We excluded men receiving antibiotics within the preceding month and those reporting a sexual partner with trichomoniasis, as routine testing for this pathogen was not performed. The study was approved by the institutional review boards of the University of Alabama at Birmingham and the JCDH.
Data were collected from the JCDH STD Clinic computerized database. Clinical data recorded for each patient included urethral symptoms (consisting of dysuria or urethral discharge), the presence or absence of urethral discharge on examination, and reported contact to a sexual partner with chlamydia, gonorrhea, or trichomoniasis. Laboratory data collected included results of NAAT, cultures, and urethral Gram stain. During the study period, NAAT was performed on urethral swabs or first void urine specimens using ligase chain reaction (Abbott Laboratories, Abbott Park, IL), polymerase chain reaction (COBAS AMPLICOR; Roche Diagnostic Systems, Inc., Branchburg, NJ), or transcription-mediated amplification (APTIMA Combo 2; Gen-Probe, Inc., San Diego, CA) assays. C trachomatis and N gonorrhoeae culture methods have previously been described.4,5 Gram stains were performed by experienced clinicians using swabs inserted 1 to 2 cm into the distal urethra and rolled onto a glass slide. Gram stains were performed on material from the first urethral swab collected and before any urine specimens were collected. A patient’s last urine void was required to be greater than 60 minutes before a swab could be collected. PMNs/oif (1000× magnification) on the Gram stain were enumerated into the following: none versus 1 to 4 versus ≥5 PMNs/oif based on an average value counted from ≥3 fields. At the JCDH STD Clinic, clinical proficiency of Gram-stain techniques by clinicians is evaluated annually by direct observation, and each month, Gram-stain results are correlated with laboratory results by trained laboratory technicians who then provide feedback to providers. To further evaluate the reliability of PMN count determination by our clinical providers, a trained observer independently read 100 Gram stains collected by the same clinicians from men with a positive C trachomatis and/or N gonorrhoeae NAAT; readings were in agreement in 98%.
Statistical analyses were conducted on Stata (StataCorp, Release 6.0, College Station, TX). Significance of relationships involving clinical characteristics, urethral PMN count distribution, and urethral pathogens were assessed through parametric (χ2 or Fisher exact tests) or nonparametric (Wilcoxon rank sum test) tests as appropriate.
Of 2629 potentially eligible men, 44 were excluded due to antibiotic use and 319 because they reported contact to a partner with trichomoniasis. The remaining 2266 eligible men, composing the study population, had a median age of 26 years (range, 13–77), and 2072 (91%) were black. Overall, 353 (16%) tested positive for C trachomatis, 462 (20%) were positive for N gonorrhoeae, and an additional 175 (8%) were coinfected with both organisms. Of these 990 infected men, 26% (n = 261) were asymptomatic, and 18% (n = 178) were both asymptomatic and without urethral discharge on examination.
Men with gonorrhea were older (median age, 25; range, 13–61) than those with chlamydia (22; range, 15–69) or coinfection (21; range, 14–49) (P <0.001). Coinfected individuals more often were black (99%) than those with chlamydia (94%) or gonorrhea (97%) alone (P = 0.006). Compared to men with chlamydia, those with gonorrhea more often reported a history of urethral discharge (88% vs. 39%) or dysuria (55% vs. 23%) and more frequently had urethral discharge on examination (88% vs. 53%) (all P <0.001) (Table 1). Frequency of symptoms or discharge in coinfected men resembled those with gonorrhea (data not shown).
Relationship of Urethral PMN Counts to Urethral Pathogens and Clinical Characteristics
Among all men, urethral Gram stain revealed ≥5 PMNs/oif in 1398 (62%), 1 to 4 in 156 (7%), and none in 712 (31%). Of 353 (16%) men with chlamydia, urethral Gram stain findings were ≥5 PMNs/oif in 291 (82%), 1 to 4 in 20 (6%), and none in 42 (12%). Of 462 (20%) men with gonorrhea, Gram stain revealed ≥5 PMNs/oif in 433 (94%), 1 to 4 in 6 (1%), and none in 23 (5%). Of 175 (8%) coinfected men, Gram stain findings were ≥5 PMNs/oif in 169 (97%), 1 to 4 in 2 (1%), and none in 4 (2%). Men with chlamydia were significantly more likely to have <5 PMNs/oif (no PMNs/oif and 1–4 PMNs/oif categories combined) or no PMNs/oif on Gram stain compared to those with gonorrhea or coinfection (P <0.001).
Men with both gonorrhea and ≥5 PMNs/oif on Gram stain more often had symptoms and/or discharge and were older than men with both chlamydia and ≥5 PMNs/oif (all P <0.001) (Table 1). In contrast, there were no significant differences in frequency of symptoms and/or discharge in men with chlamydia versus gonorrhea that had 0 to 4 PMNs/oif. Racial distribution did not differ significantly in men infected with chlamydia versus gonorrhea. Clinical characteristics of coinfected individuals resembled those with gonorrhea (data not shown).
Relationship of Clinical and Gram-Stain Findings to Therapeutic Management of Chlamydial and Gonococcal Urethritis
A distributive algorithm describing therapeutic management based on presence of symptoms, discharge, or Gram stain findings is displayed in Figure 1. Urethral symptoms (discharge or dysuria), discharge on examination, or Gram stain evidence of urethritis (≥5 PMNs/oif) was absent in 47(13%) and 22 (5%) of chlamydial and gonococcal infections, respectively (this includes those with no PMNs/oif and those with 1–4 PMNs/oif); none of the 47 chlamydial-infected individuals and only 4 of the 22 gonococcal-infected individuals (these men each reported sexual contact to a partner with gonorrhea) were treated at the time of initial examination.
Differences in Urethral PMN Counts and Clinical Characteristics by Chlamydial and Gonococcal Culture Results
A subpopulation of 104 individuals with chlamydia had both NAAT and culture performed, with 103 (99%) having a positive NAAT versus 73 (70%) being culture-positive. In the 103 chlamydial NAAT-positive individuals, there was no significant difference in the urethral PMN count distribution or frequency of symptoms in those culture-positive versus culture-negative (Table 2). Compared with chlamydia culture-negative subjects, those culture-positive somewhat more often had discharge on examination (P = 0.086). Worth noting is that in the 8 chlamydial NAAT-positive men in whom no PMNs/oif were visualized, only 4 (50%) were culture positive.
A subpopulation of 261 men with gonorrhea had both NAAT and culture performed, with 258 (99%) having a positive NAAT versus 214 (82%) being culture-positive. In contrast to men with chlamydia, there were significant differences in urethral PMN counts and clinical characteristics in the 258 gonococcal NAAT-positive men when categorized by culture results (Table 2). Gonococcal culture-positive men more often reported a history of urethral discharge or dysuria (P = <0.001 and P = 0.006), had discharge on examination (P <0.001), or had ≥5 PMNs/oif on urethral Gram stain (P <0.001). Worth noting is that in the 15 gonococcal NAAT–positive men in whom no PMNs/oif were visualized, only 2 (13%) were culture positive, and none had intracellular Gram-negative diplococci visualized on Gram stain (data not shown).
Accepted clinical diagnostic criteria for urethral inflammation (i.e., urethritis) include the presence of a urethral discharge on examination or visualization of ≥5 PMNs/oif on Gram stain from a urethral swab specimen.2 Studies performed before the availability of NAAT had found 16% to 50% of men with documented urethral chlamydial infections did not have ≥5 PMNs/oif.6–11 More recent studies using NAATs in men have confirmed that either chlamydia or gonorrhea can often be asymptomatic.12 Moreover, the higher sensitivity of NAAT, in comparison with earlier chlamydial and gonococcal tests, now enables us to better study the relationship of Gram stain findings to chlamydia and gonorrhea. In a recent study of 162 men with chlamydial urethral infection (detected by urine LCR or by urethral culture), Marrazzo et al.3 reported urine LCR afforded a substantial increase in chlamydial diagnosis over culture, and the increase was highest in men without urethral inflammation as detected by Gram stain (<5 PMNs/oif) or a leukocyte esterase test. Geisler et al.13 previously demonstrated significantly lower chlamydial inclusion-forming unit counts in chlamydia-infected men without discharge on examination or Gram-stain evidence of inflammation, perhaps further supporting the notion that the highly sensitive NAAT may be more advantageous than other chlamydial tests in patients without signs or evidence of inflammation, or in other clinical settings where a low chlamydial organism burden might be encountered. The relationship of gonococcal infection to urethral inflammation (i.e., PMNs/oif on Gram stain of urethral specimen) was not addressed in the Marrazzo et al.3 study, nor were the impact of Gram stain findings and clinical manifestations on therapeutic decisions in the study population.
We found that 18% (62 of 353) of chlamydial infections, 6% of gonorrhea (29 of 462), and 3% (6 of 175) of coinfections in men presented with 0 to 4 PMNs/oif on Gram stain. Many infections were asymptomatic and detected by NAAT but not culture. There are several possible explanations as to why NAAT may detect infection, yet there is little or no evidence of urethral inflammation. One is that there may have been false-positive NAATs. Our study was performed in men at high risk for STDs and in whom the prevalence of gonorrhea and chlamydia was high, and therefore false-positive NAATs are unlikely. In populations with a lower prevalence of infection, the positive predictive value of NAAT may be reduced.14 However, in such populations, the proportions of infected men with asymptomatic urethral infections would also likely be higher than in this study and urethral Gram stain might be performed less often, offsetting the impact of reduced positive predictive value. Another explanation is a false-negative Gram stain test for inflammation. While differences in time since micturition and sample collection and also observer variation (in PMN enumeration) could have influenced detection and categorization of PMNs/oif in our study, the large sample size, the requirement for the last void to have occurred more than 60 minutes before specimen collection, and the provider experience in Gram-stain interpretation would reduce the likelihood of inaccurate Gram-stain findings and the impact of such influences on our study findings. Finally, it is possible that some individuals may have just been recently exposed to chlamydial organisms at the time of testing, and the inoculum was either in the process of being cleared by the host immune system or establishing infection that would later yield evidence of urethral inflammation that could be detected if repeat examination was performed at a later date. Perhaps other measures of inflammation, such as inflammatory mucosal cytokines, may provide earlier evidence of inflammation before PMNs are recruited and detected.
There are diagnostic and management implications of the findings from our study, which is the largest study to date assessing the relationship of urethral inflammation (i.e., PMNs/oif on Gram stain of a urethral specimen) with chlamydial and gonococcal urethral infection detected by NAAT. Our study demonstrated men with chlamydial infection are usually asymptomatic and often without discharge or ≥5 PMNs/oif on Gram stain, and hence the clinical presentation is sometimes less helpful in guiding therapeutic decisions, emphasizing the need for chlamydial diagnostic testing to be performed to detect this infection; as demonstrated in our study and others, NAAT will detect more chlamydial infections in asymptomatic men than culture. Most men with gonococcal urethral infection will have symptoms, discharge, and/or ≥5 PMNs/oif on Gram stain, and culture will detect most of these infections. However, our study demonstrated that gonococcal-infected men that are culture-negative are often without symptoms or discharge and have <5 PMN/oif (Table 2), supporting the notion that NAAT would be more advantageous for detecting gonorrhea in this subpopulation as well. We also found that in most instances where men are asymptomatic, without discharge, and without ≥5 PMNs oif on Gram stain, therapy for chlamydia or gonorrhea would not likely be administered unless patients are identified as a sexual contact to an infected partner. The finding that 4 men with such a clinical presentation were empirically treated as contacts to gonorrhea and indeed tested positive for gonorrhea justifies the practice of epidemiologic treatment, as individuals who are contacts might have a lower burden of organisms when they present for testing and not have clinical symptoms or signs at that stage of their infection. Overall, 13% of chlamydial and 4% of gonococcal infections initially went untreated in our study, placing sexual partners at risk for infection and both patients and their partners at risk for complications.
A limitation of our retrospective study was that we could not exclude individuals with other potential pathogens, such as Mycoplasma or Ureaplasma species, that were not routinely tested for in clinical practice, and such organisms could influence Gram-stain findings in patients with or without chlamydia or gonorrhea. While the role for Ureaplasma urealyticum in urethral infection has been difficult to establish, there is stronger evidence supporting Mycoplasma genitalium as an etiologic agent for urethral infection.15M genitalium urethral infection has been reported to more often present with urethral symptoms when compared with chlamydial infection.16 Our large sample size and the small percentage of chlamydia-infected individuals with symptoms may decrease the likelihood the presence of M genitalium would have significantly influenced the associations we observed.
In conclusion, we have shown that chlamydial or gonococcal infections may have little or no Gram-stain evidence of urethral inflammation. In such circumstances, men are more likely to be asymptomatic, without a discharge finding, and culture is often negative. NAAT may be a better screening tool than culture for asymptomatic men. NAATs provide advantageous tools for bacterial STD control efforts focused on men both because they can be performed without invasive specimen collection and because of their improved sensitivity.
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