Viral and bacterial aetiologies of male urethritis: findings of a high prevalence of Epstein

Abstract

Male urethritis is one of the most common sexually transmitted infections (STIs). However, the aetiology is still unclear in many cases. In this study the prevalences of Epstein–Barr virus (EBV), herpes simplex virus type 1 (HSV-1), HSV-2, cytomegalovirus (CMV), adenovirus, Chlamydia trachomatis, Mycoplasma genitalium and Ureaplasma urealyticum (including subtyping) were investigated. Samples from 112 male STI attendants with microscopically verified urethritis and from a control group of 103 men without clinical or microscopic signs of urethritis were analysed. Prevalences in the urethritis group compared with the controls were as follows: EBV 21%, 6% (P < 0.01); C. trachomatis 15%, 3% (P < 0.01); M. genitalium 6%, 1% (P = 0.067) and U. urealyticum 10%, 10% (ns). The results for HSV-1, HSV-2, CMV and adenovirus were negative in patients, and therefore not analysed in the controls. EBV was shown to be an independent predictor of urethritis and may play a role in its pathogenesis.

Keywords

urethritis (non-specific)Europe urethritis (viral)urethritis (bacterial)

INTRODUCTION

Urethral inflammation (urethritis) is common among male patients attending sexually transmitted infection (STI) clinics. Patients can present with symptoms like discharge, dysuria or urethral pruritus, but asymptomatic cases are common.¹ Recognized causes of urethritis are Neisseria gonorrhoeae and Chlamydia trachomatis. Mycoplasma genitalium is another established cause of non-gonococcal, non-chlamydial urethritis (NGNCU),² and Ureaplasma urealyticum (biovar 2, subtype 1) has also been implicated as an aetiologic agent in some studies, while others have not been able to confirm this association.^3,4 Even though defined pathogens can be found in many cases of urethritis, the aetiology is still unknown in the majority of cases of NGNCU. Few studies have addressed the role of viruses in the pathogenesis, and the primary aim of this investigation was to elucidate the role of viral microorganisms as possible aetiological agents of male urethritis.

METHODS

Study population

This study was performed among male attendees at the STI clinics of Sahlgrenska University Hospital, Södra Älvsborgs Hospital and Skövde Kärnhospital during periods from 2004 to 2007. Patients were not included when the clinic was too busy or when other than the authors were working. Initially 124 patients were included, but 12 of the samples were lost for analysis. As many as 70 patients (63%) reported earlier episodes of urethritis. Twenty-four patients had been treated with antibiotics for urethritis within the previous three months.

A questionnaire was used to record symptoms, sexual history and demographics. The inclusion criterion for patients was microscopic urethritis, defined as ≥5 polymorphonuclear leukocytes (PMNL) per high power field (hpf) (×1000 magnification) observed in ≥5 fields in a methylene-blue-stained smear of urethral secretion, collected with a plastic loop. As controls, 103 patients attending the STI clinics for reasons other than urethritis were included. Inclusion criteria for the controls were no microscopic signs of urethritis (≤2 PMNL/hpf) and no symptoms consistent with urethritis. The level of ≤2 PMNL/hpf was set in order to exclude patients with unclear microscopic findings. Everyone included in the study was offered a test for human immunodeficiency virus (HIV). None of the study subjects were on antibiotics or had visual lesions of genital herpes or balanitis involving meatus when entering the study. Demographic data are presented in Table 1.

Table 1

Demographic data for patients and controls

	Urethritis group (112)	Control group (103)	P value
Age (median)	28 years (range 20–48)	30 years (range 20–56)
No. of partners last 6 months	2.3 (0–10 partners)	2.3 (0–6 partners)
Sexual preference
Heterosexual	100/101	103/103
Bisexual	1/101
History of earlier STI	52/99 (53%)	31/102 (30%)	P < 0.01
Chlamydia	40/99 (40%)	20/101 (20%)
Condyloma	19/99 (19%)	8/101 (8%)
Genital herpes	10/99 (10%)	5/101 (5%)
M. genitalium	1/99 (1%)	0
≥1 diagnosis	18/99 (18%)	3/101 (3%)
Condom
Always	23/100 (23%)	21/101 (21%)	ns*
Sometimes	69/100 (69%)	75/101 (74%)	ns
Never	8/100 (8%)	5/101 (5%)	ns
Antibiotics last 3 months	24/93 (26%)	5/98 (5%)
Cause for visit
Discharge/dysuria	98/112	0
No registered cause	0	7/103
STI ‘check-up’	10/112	85/103
Condyloma	1/112	2/103
Balanoposthitis	1/112	5/103
Partner notification	2/112	0
Partner symptom	0	4/103
Urethral symptoms	98/112 (88%)	0/103
Discharge + dysuria	43/112 (38%)	0
Discharge	14/112 (13%)	0
Dysuria	41/112 (37%)	0
PMNL/hpf
≤2	0	103/103
5–9	63/112 (56%)	0
≥10	49/112 (44%)	0

STI = sexually transmitted infection; PMNL = polymorphonuclear leukocyte; hpf = high-power field

*ns = non-significant

Clinical specimens

In the urethritis group, samples for analysis of Epstein–Barr virus (EBV), herpes simplex virus type 1 (HSV-1), HSV-2, cytomegalovirus (CMV), adenovirus and U. urealyticum biovar 2 (subtyped to no. 1, 2 or 3) were taken with a thin cotton-tipped swab from the urethra and transported in a tube with 1 mL of sterile NaCl. A first-void urine (FVU) sample (incubation time in the bladder ≥1 hour) was collected for the analyses of C. trachomatis and M. genitalium.

In the control group, urethral specimens were collected as above for analysis of EBV and U. urealyticum. Since no samples from the urethritis group were positive for HSV-1, HSV-2, CMV or adenoviruses, these analyses were not performed in the control group. FVU samples were analysed using polymerase chain reaction (PCR) of C. trachomatis and M. genitalium.

A subset of samples was tested for the β-globin gene to ensure that the samples were suitable for DNA amplification. An analysis of phocid herpesvirus type 1 (PhHV-1) was used as an internal control, showing that the viral DNA extraction procedure was adequate. None of the included patients or controls had Gram-negative diplococci in their urethral smear and testing for gonococcal infection was not carried out. Testing for Trichomonas vaginalis was not performed due to the low prevalence in our population. All specimens were kept frozen at −70°C until analysed.

Microbiological methods

PCR detection of EBV, HSV-1, HSV-2, CMV and adenovirus DNA

DNA extraction from 200 μL urethral specimens was done in the automated system MagNa Pure LC (Roche Diagnostics, Mannheim, Germany) using the DNA Isolation Kit І and the high performance extraction protocol. Real-time PCR (RT-PCR) (TaqMan, Applied Biosystems, Foster City, CA, USA) was used for detection of HSV-1, HSV-2, human β-globin and PhHV-1 and for detection and quantification of CMV, EBV and adenovirus. The method, primers and probe used for detection of HSV-1/2 were published in 2005⁵ and Heim⁶ described primers and probe sequences used for the detection of adenovirus. The CMV-PCR was employed as described earlier.⁷ Primers and probe that target the non-glycosylated membrane protein BNRF p143 were used for the analysis of EBV.⁸ As internal controls, PhHV-1 primers and probe were adapted from the work by Niesters.⁹ The virus stock was diluted 1000 times in phosphate-buffered saline, in accordance with the manufacturer's instruction, in order to obtain TaqMan PCR cycle threshold values around 28 cycles. Tucker et al. ¹⁰ described primer and probe sequences used for the β-globin analysis.

Nucleic acid amplification test detection of C. trachomatis

C. trachomatis was detected by the DNA amplification technique strand displacement amplification using BD ProbeTec ET System C. trachomatis amplified DNA assay (Becton Dickinson Diagnostic Systems, Franklin Lakes, NJ, USA) as recommended by the supplier. This test also detects the mutant variant of C. trachomatis.

PCR detection of M. genitalium and U. urealyticum

Urine specimens for identification of M. genitalium and U. urealyticum by PCR were prepared as described earlier.¹¹ Swab specimens were prepared using the Respiratory Specimen Preparation Kit (Roche Diagnostics, Scandinavia AB, Bromma, Sweden) according to the manufacturer's instructions. Primers used for detection of M. genitalium were described by Jensen et al. ¹² The method and primers used for detection of ureB in the urease gene cluster of U. urealyticum were published in 2005.¹³ Subtype determination of U. urealyticum was performed by sequencing of the 5′ end of the gene coding for the multiple-banded antigen.³ The nucleotide sequences of PCR products were determined by ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Stockholm, Sweden) with ABI PRISM BigDye Terminator Cycle Sequencing Kit, version 1.1 (Applied Biosystems). After DNA sequence editing, the GenBank BLAST programme (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for sequence comparisons and determination of subtype.

Statistical analysis

Statistical analyses were performed using SPSS software version 15 (SPSS Inc, Chicago, IL, USA). Clinical and laboratory findings in cases were compared with controls using Fisher's exact test. Logistic regression was used to control for confounding factors, including co-variations between pathogens.

Ethical approval

This study was approved by the Ethics Committee of the Medical Faculty of the University of Göteborg. All patients and controls provided written informed consent.

RESULTS

A statistically significant difference in prevalence between patients and controls was seen for EBV, but HSV-1, HSV-2, CMV and adenovirus were negative in all patients and therefore not analysed in the control group. A significant difference was seen for C. trachomatis, but for M. genitalium this difference was not significant (P = 0.067). U. urealyticum was as common in the control group as in the patient group. In total, 42/112 patients were positive in any of the tests, and of these 17 were positive in two tests. Results are shown in Table 2.

Table 2

Results for patients and controls

	Urethritis group	Control group	P value
EBV	24/112 (21%)	6/103 (6%)	P < 0.01
EBV + C. trachomatis	10/24	1/6	ns
EBV + M. genitalium	1/24	0/6	ns
EBV + U. urealyticum	4/24	0/6	ns
HSV-1	0	Not done
HSV-2	0	Not done
CMV	0	Not done
Adenovirus	0	Not done
C. trachomatis	17/112 (15%)	3/103 (3%)	P < 0.01
M. genitalium	7/112 (6%)	1/103 (1%)	P = 0.067
U. urealyticum	11/112 (10%)	10/103 (10%)	ns
Subtype 1	2/11	3/10
Subtype 2	4/11	4/10
Subtype 3	1/11	0
Indeterminate	4/11	3/10

EBV = Epstein–Barr virus; HSV = herpes simplex virus; CMV = cytomegalovirus

Samples positive for EBV (24/112) had a mean number of 1343 copies/mL (range 133–11,472). Of these, nine were positive only for EBV (mean value 1594 copies/mL), 10 were also positive for C. trachomatis (1612 EBV copies/mL), four also for U. urealyticum (355 EBV copies/mL) and one also for M. genitalium (350 EBV copies/mL). EBV was found to be independently correlated to urethritis (P = 0.008), using logistic regression analysis. In this analysis co-infected EBV cases were included, but not quantification of EBV. Previous chlamydia infection (P = 0.001) and ongoing chlamydia infection (P = 0.019) were also independently associated with urethritis. For distribution of pathogens according to microscopic findings, see Table 3.

Table 3

Distribution of pathogens according to microscopic findings

Microscopy	C. trachomatis (n = 20)	M. genitalium (n = 8)	U. urealyticum (n = 21)	EBV (copies/mL) (n = 30)	EBV + C. trachomatis
≤2 PMNL/hpf (n = 103)	3	1	10	6 (290)	1/6
5–9 PMNL/hpf (n = 63)	10	5	6	9 (1756)	5/9
≥10 PMNL/hpf (n = 49)	7	2	5	15 (1096)	5/15

PMNL = polymorphnuclear leukocyte; hpf = high-power field

In the control group, 19/103 were positive in any test and of these one participant was positive in two tests. EBV-positive samples (6/103) in the control group had a mean value of 290 copies/mL (range 200–500). Of these, one sample was also positive for C. trachomatis (200 EBV copies/mL). Comparison of the number of solely EBV-infected samples, excluding co-infected individuals, in the urethritis group (9/112, mean value 1594 copies EBV/mL) and in the control group (5/103, mean value 308 copies/mL) did not show a statistic difference (P = 0.41).

All samples tested for β-globin and PhHV-1 were positive. All HIV tests were negative (138/215). Reasons for not testing were that the patients thought their risk for HIV was low, or they had a recent negative test or a fear of taking blood samples. Men who have sex with men were not included in the study, since they attend a special clinic in our hospital.

DISCUSSION

In this study we found an independent association between male urethritis and EBV, but results for other viral organisms were negative. As expected, C. trachomatis was significantly more common in the group with microscopic urethritis compared with the control group. A history of earlier STI was more often reported in the group with urethritis, but the use of condoms and the reported number of partners during the previous six months did not differ.

The main transmission route for EBV is saliva, and EBV commonly spreads during childhood and in young adults. EBV primarily infect B lymphocytes, but can also infect epithelial cells. Evidence for EBV in epithelial cells of the uterine cervix has been found in cervical washings, raising the question of sexual transmission of EBV.¹⁴ Further, studies from our clinic demonstrated EBV DNA in epithelial cells also from penile skin and vulvar mucosa.¹⁵ In a study of men with urethral discharge and gonococcal infection, excretion of EBV was detected in cell-free filtrates of the discharge, but the authors could not exclude the possibility that this represented B-cell-associated virus.¹⁶ The finding in the current study of EBV DNA in 21% of the patients with urethritis, compared with only 6% in the control group, raises many questions. EBV was commonly seen in chlamydia-infected individuals (10/24), indicating that EBV might be activated as a co-pathogen. But even if the activation of EBV as a co-pathogen or the presence of PMNL-associated EBV cannot be excluded, quantification of EBV DNA showed a higher mean number (1343 copies/mL) in patients compared with controls (290 EBV copies/mL). Also, comparing the amount of EBV in the group of urethritis between co-infected individuals (mean value 1193 EBV copies/mL) and those infected with only EBV (mean value 1594 copies/mL) did not show that co-infection led to higher quantities of EBV. Additional studies are needed to further investigate genital EBV infection and analysis of EBV RNA may give more information.

In some studies of HSV, urethritis has been shown to be the sole symptom of genital herpes.¹⁷ However, our study could not confirm this association. On the other hand, HSV causing urethritis with concurrent herpetic lesions or meatitis⁴ is more common, and since herpetic lesions and meatitis were exclusion criteria in our study, this might explain why no HSV-positive samples were found. Adenovirus detected by PCR was seen in 4% in a study of urethritis, and the conclusion was that adenovirus is uncommon, but should be considered especially in patients with urethritis and concomitant meatitis.^4,18 As for HSV, our exclusion criteria could explain the negative adenovirus results. Another cause could be that the inclusion criteria of ≥5 PMNL/hpf is better suited to bacterial infections and one hypothesis could be that cases of urethritis caused by viruses might be found more easily when mononuclear cells are seen, rather than PMNL. Microscopic findings of mononuclear cells were not included in our study, but this hypothesis might also explain why no cases of CMV were found. As shown by Bradshaw et al. ⁴ genital infections with adenoviruses and HSV-1 are associated with oral sex. Data on oral sex were not available in our study population, but we know from earlier studies that this is a common practice in our STI population.¹⁹

The prevalences of C. trachomatis and M. genitalium were lower than in other studies of urethritis. This may be explained by the inclusion of a high number of patients with recurrent urethritis and recent antibiotic treatment (mainly tetracycline). The low prevalence of C. trachomatis is not due to problems in diagnosing the mutant variant, since this was detected by the PCR method employed in our study.

U. urealyticum was as common in the control group as in the patient group with urethritis (10/103 versus 11/112). The subtypes were similarly distributed in both groups. Some studies have found a link between U. urealyticum and urethritis, mainly subtype 1, while others have not.^3,4 Further studies with a higher number of patients are needed to determine whether there is a definite correlation between urethritis and specific subtypes.

In conclusion, the high rate of samples positive for EBV is a novel finding, and EBV was found to be independently correlated to urethritis. Future studies are needed in order to clarify the role of EBV as a pathogen in urethritis.

Footnotes

ACKNOWLEDGEMENTS

We thank the staff of the Dermatology Department of Skövde Kärnhospital for data collection, Zoreh Sadegsadeh for excellent technical assistance and Martin Gillstedt for performing the statistical analysis. The work was supported by the Medical Society of Göteborg, the LUA Foundation at Sahlgrenska University Hospital and the Swedish Research Council, grant no. 11225 and GSK scholarship in venereology.

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Viral and bacterial aetiologies of male urethritis: findings of a high prevalence of Epstein–Barr virus

Abstract

Keywords

INTRODUCTION

METHODS

Study population

Clinical specimens

Microbiological methods

PCR detection of EBV, HSV-1, HSV-2, CMV and adenovirus DNA

Nucleic acid amplification test detection of C. trachomatis

PCR detection of M. genitalium and U. urealyticum

Statistical analysis

Ethical approval

RESULTS

DISCUSSION

Footnotes

ACKNOWLEDGEMENTS

References