Ureaplasma parvum and Mycoplasma genitalium are found to be significantly associated with microscopy-confirmed urethritis in a routine genitourinary medicine setting

Abstract

Summary

Inflammation of the urethra defined by an excess of polymorphonuclear leukocytes in the absence of sexually transmitted Chlamydia trachomatis and Neisseria gonorrhoeae is called non-chlamydial non-gonococcal urethritis (NCNGU). Although Mycoplasma genitalium is now recognised as causing a sexually transmitted infection, the clinical significance of the other Mollicute species is less clear. This study used specific real-time quantitative polymerase chain reaction assays to detect and quantify four Mollicute species, M. genitalium, M. hominis, Ureaplasma urealyticum and U. parvum, in urine specimens from men with and without NCNGU. A total of 165 urine specimens from male patients attending a genitourinary medicine clinic were eligible for the study, with microscopy-confirmed (≥5 polymorphonuclear leukocytes in urethral swab) NCNGU in 75 (45.5%) and non-confirmed NCNGU in 90 (54.5%). Chi-squared statistical analysis indicated a significantly higher prevalence of U. parvum (17.3% vs. 5.6%; p = 0.03) and M. genitalium (12% vs. 0%; p < 0.001) in NCNGU. In a subset analysis, M. genitalium was also significantly (p = 0.03) higher in men who have sex with men (MSM; 13.5%) compared to non-MSM (3.1%). No significant associations were reported for U. urealyticum and M. hominis. In conclusion, this study supports a clinically significant role in NGNCU for both U. parvum and M. genitalium.

Keywords

Non-gonococcal non-chlamydial urethritis real-time quantitative PCR sexually transmitted infection men

Introduction

Inflammation of the urethra, urethritis, is characterised by urethral symptoms including painful urination, discharge and irritation; symptoms may be absent or non-specific.¹ Urethritis is associated with sexually transmitted infections (STIs), with Neisseria gonorrhoeae and Chlamydia trachomatis the most commonly recognised bacteria causally linked with the condition.¹ Significant proportions of men attending STI clinics who have microscopic demonstration of an excess of polymorphonuclear leukocytes (PMNLs) in a discharge or first void urine (FVU) test negative for both pathogens and are classified as having a non-chlamydial non-gonococcal urethritis (NCNGU).^1,2 Recurrent or persistent symptomatic urethritis after treatment can be a management problem when following an empirical protocol.^3,4 Persistence of Mycoplasma genitalium and Ureaplasma species post treatment makes a role in recurrent urethritis plausible but not certain.^4–7 A more inclusive testing regimen to identify causative organisms could provide objective data both pre- and post treatment and may influence further management decisions. Employment of nucleic acid amplification tests (NAATs) to clinical specimens has improved our understanding of the role of Mollicutes in NCNGU.^8–11 Even though M. genitalium is now a recognised cause of NCNGU in men, routine testing of this organism is not performed in most clinical settings. Furthermore, much of the published literature is ambiguous regarding the role of other genital Mollicutes in NCNGU.^3,4,12–16 Identification of genital Mycoplasma and Ureaplasma species in NCNGU, where 20–50% of cases are currently thought to be missed,¹ would be useful for both patient management decisions and supporting research into the condition.

Extended testing for NCNGU is now being provided outside genitourinary medicine (GUM) clinics leading to self-referral and a need for better understanding of these infections by clinicians. This prompted the current study to assess the prevalence of M. genitalium, M. hominis, U. urealyticum and U. parvum in men with and without NCNGU. Target-specific real-time quantitative polymerase chain reaction (qPCR) assays were applied to urine specimens from symptomatic men. NCNGU was confirmed in patients using the British Association for Sexual Health and HIV (BASHH) gold standard of PMNL counts in urethral discharges screening negative for both N. gonorrhoeae and C. trachomatis.

Methods

Study population

Urine specimens were collected from symptomatic men attending a local GUM clinic over a one-year consecutive period; specimens positive for C. trachomatis and/or N. gonorrhoeae (CT/NG) were excluded. BASHH 2014 guidelines¹⁷ outline the current gold standard diagnosis for NCNGU, which is the presence of ≥5 PMNLs/high power field (hpf) in a urethral smear in the absence of C. trachomatis and N. gonorrhoeae; therefore, only men with a concurrent urethral smear sent for microscopy examination of PMNLs were included in this study. Examination for PMNLs is performed on urethral smears from symptomatic men attending GUM, whereas asymptomatic men submit only a urine sample for routine STI testing. Men with ≥5 PMNLs/hpf were regarded as NCNGU and men with <5 PMNLs/hpf as without NCNGU. Where a rectal swab was also sent for routine CT/NG testing, then an inference was made to categorise them as ‘men who have sex with men’ (MSM) and non-MSM for men without a rectal swab.

Specimen collection

Urine specimens were collected using the Cobas® PCR Urine Sample Kit (Roche Molecular Diagnostics, Mannheim, Germany) and transported to the laboratory in the collection media provided with the kit and stored at 4°C for no longer than one week. Urethral swabs were collected on a cotton-tipped swab passed into the distal urethra, smeared onto a glass slide, covered and sent to the routine bacteriology laboratory for analysis.

Gram staining

Urethral smeared slides were heat fixed and gram stained, and the PMNLs per hpf at 1000× magnification were recorded. Urethritis was defined if ≥5 PMNLs/hpf were present (averaged over five fields with the highest concentration).

Molecular testing

Urine specimens were subjected to Cobas® 4800 CT/NG testing (Roche Molecular Diagnostics, Mannheim, Germany). Nucleic acid extracts negative for CT/NG were collected in batches on a fortnightly basis after Cobas® 4800 CT/NG testing and subject to four real-time qPCR assays targeting U. urealyticum, U. parvum²³ and M. hominis²⁴, M. genitalium²⁵ using LightCycler 480® instrumentation (Roche Diagnostics, Mannheim, Germany). Clinical and analytical validation has been completed in our laboratory for each of the real-time qPCR assays. Logarithmic dilutions of gene-specific plasmid standards tested against each qPCR assay demonstrated limits of detection of less than 54 gene copies per 2 µl, and no cross reactivity was observed when the four qPCR assays were tested against a panel of nucleic acid extracts from 40 different organisms. All assays in this study were carried out using 1X Platinum® Quantitative PCR SuperMix-UDG (Life Technologies, Paisley, UK). Final working concentrations of reagents in the qPCRs assays were as follows: 0.4 µM forward and reverse primer, 0.1 µM TaqMan® probe, 0.2 µg ml⁻¹ BSA (Thermoscientific, Epsom, UK) and 4 mM MgCl₂ (Life Technologies, Paisley, UK). Final reaction volumes of 10 µl comprising 2 µl nucleic acid extract and 8 µl mastermix were used with the following PCR cycling conditions: 95°C for 5 min, followed by 45 cycles of 95°C for 15 s and 59°C for 45 s. MS2 internal control was included in each real-time PCR run to monitor PCR inhibition and DNA extraction. Results were analysed using LightCycler® 480 software, version 1.5 (Roche Diagnostics, Mannheim, Germany) and recorded as cycle threshold (C_T) value, any reactions which failed to produce a C_T value after 45 cycles were recorded as negative. A gene-specific plasmid standard of 10⁵ gene copies ml⁻¹ was included to allow quantification against an internally stored calibration curve and recorded as genome copy number ml⁻¹.

Statistical analysis

Statistical analyses were performed using SPSS 19.0 (SPSS Science, Chicago, IL, USA) statistical package. Continuous variables were expressed as means and categorical variables were expressed as counts and percentages. Chi-squared analysis was used to compare U. urealyticum, U. parvum, M. hominis and M. genitalium prevalence in urine samples between (a) men with and without NCNGU and (b) MSM and non-MSM. Fisher’s exact test was used where 20% of cells contained fewer than five elements. Independent t-test analysis was performed to compare the bacterial loads of the four organisms between men with and without NCNGU. A p value <0.05 was regarded as statistically significant.

Ethical approval

All specimens used in this study were anonymised residual specimens from Microbiology laboratories at Belfast Health and Social Care Trust; the specimens were collected and processed under research ethics approval from Northern Ireland REC Committee 1, reference number 10/NIR01/20.

Results

Urethral PMNL count

A total of 165 symptomatic men were included in the study: 75 (45.2%) had microscopy-confirmed (≥5 PMNLs) NCNGU, and 90 (54.2%) were microscopy non-confirmed (<5 PMNLs) NCNGU.

Comparison of men with and without NCNGU

U. urealyticum, U. parvum, M. genitalium and M. hominis were detected in 20 (12.1%), 18 (10.9%), 9 (5.5%) and 5 (3.0%) of the men studied, respectively, the remaining 113 (68.5%) men had no Mollicute infection detected. There was a higher prevalence of U. urealyticum, U. parvum and M. hominis in men with NCNGU (16.0%, 17.3% and 4.0%, respectively) compared to those without NCNGU (8.8%, 5.6% and 2.2%, respectively). M. genitalium was detected in 9 (12%) men with NCNGU and was not detected in men without NCNGU. Comparisons of organism prevalence between men with and without NCNGU are summarised in Table 1 and Figure 1. The difference in M. hominis and U. urealyticum prevalence between the two groups did not reach statistical significance. The higher U. parvum and M. genitalium prevalence in men with NCNGU were statistically significant (p = 0.03 and <0.001, respectively). An odds ratio of 3.6 (95% C.I. 12–10.5) was calculated for U. parvum in men with NCNGU.

Table 1.

Comparison of Mollicute prevalence in urine samples from men with and without NCNGU using Chi-squared statistical analysis.

	Total number of positives (%)		p	Odds ratio	95% C.I.
	With NCNGU (n = 75)	Without NGNCU (n = 90)	p	Odds ratio	Lower	Upper
U. urealyticum	12 (16%)	8 (8.8%)	0.24 NS	2.0	0.7	5.1
U. parvum	13 (17.3%)	5 (5.6%)	0.03*	3.6	1.2	10.5
M. hominis	3 (4%)	2 (2.2%)	0.66 NS	1.8	0.3	11.2
M. genitalium	9 (12%)	0	<0.001*	N/A	N/A	N/A

Significance <0.05.Mollicute load in men with and without NCNGU.

Figure 1.

Mollicute prevalence in men with and without non-chlamydial non-gonococcal urethritis (NCNGU).

The loads of U. urealyticum, U. parvum and M. hominis detected in men with confirmed NCNGU compared to men with non-confirmed NCNGU did not reach statistical significance.

Comparison of loads between the two groups are summarised in Table 2. M. genitalium load comparison was excluded from analysis since it was only detected in men with NCNGU.

Table 2.

Comparison of Mollicute load in men with confirmed and non-confirmed urethritis.

	Mean gene copy number ml⁻¹		p ^a	Mean difference	95% C.I.
	With NCNGU (n = 75)	Without NCNGU (n = 90)	p ^a	Mean difference	Lower	Upper
U. urealyticum	7.7 × 10⁴	4.3 × 10⁵	0.48	−0.22	−4.4	0.9
U. parvum	1.9 × 10⁵	4.3 × 10⁵	0.69	0.22	−1.4	0.9
M. hominis	1.52 × 10⁶	3.1 × 10⁸	0.35	−2.1	−3.9	8.1

Comparison of log₁₀ transformation of mean loads.

Comparison between MSM and non-MSM

The prevalence of M. genitalium was significantly (p = 0.03) higher in MSM (13.5%) compared to non-MSM (3.1%). The prevalence of U. parvum was higher in non-MSM (13.3%) compared to MSM (2.7%) and approached statistical significance (p = 0.08). The prevalence of U. urealyticum in MSM and non-MSM (13.0% vs. 11.7%) was similar; M. hominis was only detected in non-MSM (3.9%). Statistical comparisons of the four Mollicute bacteria between the two groups of men are summarised in Table 3.

Table 3.

Mollicute prevalence comparison in men who have sex with men (MSM) and non-MSM.

	MSM (%) (n = 37)	Non-MSM (%) (n = 128)	p	O.R. (95% C.I.)
U. urealyticum	5 (13.0)	15 (11.7)	0.77	1.18 (0.40–3.49)
U. parvum	1 (2.7)	17 (13.3)	0.08	0.17 (0.02–1.34)
M. hominis	0	5 (3.9)	0.59	0.96 (0.93–0.99)
M. genitalium	5 (13.5)	4 (3.1)	0.03	4.8 (1.23–19.08)

Discussion

The need to understand the role of genital Mycoplasma and Ureaplasma species in symptomatic NCNGU is increasing. While M. genitalium is now a recognised cause of 15–20% of NCNGU cases,^4,12–16 the role of Ureaplasma species and M. hominis is less certain.^18–22 Genital Mycoplasma and Ureaplasma species are no longer difficult to detect due to the advent of NAATs,^23–25 providing the opportunity to improve the evidence base of their role in urethral inflammation. This study used optimised real-time qPCR assays to detect and quantitate four genital Mollicute species in urines from men with and without microscopy-confirmed urethral inflammation. A total of 165 residual anonymised FVUs negative for C. trachomatis and N. gonorrhoeae from symptomatic male patients in a GUM setting were tested. Men were dichotomously categorised as NCNGU (54.5%) and without NCNGU (45.5%) using the current gold standard criteria, which clearly outlines ≥5 PMNLs/hpf in the anterior urethra confirms urethritis¹⁷ and the prevalence of each genital Mollicute in each category is compared. U. urealyticum (12.1%) had the highest prevalence followed in descending order by U. parvum (10.9%), M. genitalium (5.5%) and M. hominis (3.0%). The increased prevalence of U. parvum (17.3% vs. 5.6%) and M. genitalium (12% vs. 0%) in NCNGU was statistically significant; the prevalence of U. urealyticum and M. hominis was not significant. There was also no significant difference in the respective loads between men with NCNGU and without NCNGU.

M. genitalium was not detected in specimens from the 90 men without NCNGU. The clear association (p < 0.001) of M. genitalium presence in patients with high urethral PMNL count strongly suggests a causal role in NCNGU in keeping with the current literature.^18,26 The significant association (p = 0.03) of U. parvum in specimens from patients with a high urethral PMNL count would suggest a potential association with NCNGU and the need for further powered studies with appropriate microbiological stratification to confirm the association.

While the case for M. genitalium as one cause of NCNGU is accepted,²⁷ the results for U. parvum and U. urealyticum were unexpected and less in keeping with the published literature. The majority of manuscripts link U. urealyticum with NCNGU and suggest U. parvum as a non-pathogenic commensal.^{8,18–20,28–30} Despite the recent separation of U. urealyticum biovars into U. urealyticum (previously biovar 2) and U. parvum (previously biovar 1),³¹ studies often report them as a single species, lacking the discrimination needed to assign clinical significance.^4,8,19,30,32 At the genus level, Ureaplasma species have been reported to induce significant inflammatory responses in non-genital sites, including foetal membranes, umbilical cord and early onset sepsis.^33–37 When reported at a species level, U. parvum rather than U. urealyticum has been associated with intrauterine inflammation.³⁵ The current findings suggest that U. parvum may act as a low-grade pathogen able to induce inflammation in the male urethra. Further appropriately powered studies against the current gold standard of PMNL urethral infiltration, often omitted from studies,¹⁷ would be needed to confirm the consistency of this association.

While M. genitalium was only detected in NCNGU, the loads of the other three Mollicutes were lower in men with NCNGU compared to men without NCNGU, but none were statistically significant; these lower loads may reflect prior antibiotic exposure. By contrast, Shimada et al.²⁹ reported significantly higher loads of U. urealyticum in NCNGU, also defined by raised PMNLs counts. Although all specimens subjected to testing in the study were recorded as FVU, it was impossible to confirm this, as sample collection was not directly observed. However, the categorical detection of M. genitalium only in NCNGU supported the classification into with and without NCNGU based on PMNL infiltration. Standardisation of specimen collection and access to an antibiotic history would be important for inferences on load and inflammation in future studies.

The current standard of care for clinical management of patients with NCNGU does not require laboratory confirmation, and empirical treatment is directed by guidelines principally from BASHH.³ However, there is a growing concern this is encouraging antibiotic resistance, supported by confirmation of the rise in macrolide and quinolone antibiotics resistance.^3,38 Single-dose azithromycin has a low cure rate in M. genitalium-positive patients and drives M. genitalium resistance.^3,38

Men in this study who had a rectal swab submitted for STI testing were categorised as MSM, allowing additional stratification of the study cohort into MSM and non-MSM for the purposes of analysis, in line with the local protocol of screening a rectal swab in MSM at each visit. Thirty-seven (22.4%) of men in the study were categorised as MSM while 128 (77.6%) were categorised as non-MSM. The higher prevalence of U. urealyticum, U. parvum and M. hominis in non-MSM could be in keeping with exposure to them in the female genital tract where they are commonly found; Ureaplasma species are reported to be present in 40–80% of sexually active women,³⁹ and so the finding of a significant association of U. parvum with non-MSM is therefore not surprising. The high colonisation rate of U. parvum in females³⁹ would suggest that the normal vaginal microflora could act as the source of infection in non-MSM. This would make contact tracing problematic if testing were to become routine. The lower frequency of U. parvum and M. hominis in MSM supports the theory that transmission may be more common in heterosexual couples.

The prevalence of M. genitalium and U. urealyticum was higher in urine samples from MSM but only M. genitalium reached statistical significance (p = 0.001). Studies are limited worldwide in assessing the prevalence of M. genitalium in the MSM population.⁴⁰ In Northern Ireland, figures were unavailable before this study as routine testing is not undertaken for M. genitalium in either the MSM or non-MSM populations. However, there is a growing body of evidence for its role in NCNGU.^14,41,42 M. genitalium has been detected in men with HIV and its adherence to HIV-infected cells has demonstrated an increase in virus release^26,43,44; thus, it is possible that undiagnosed M. genitalium infection could influence the risk of acquiring and transmitting HIV infection.^45,46 Data demonstrated in this study confirm a role for M. genitalium in NCNGU and a higher prevalence in MSM. Further research is clearly needed of the significance of these findings and in particular whether the high rates of undiagnosed infection may influence HIV transmission in MSM. In conclusion, M. genitalium and U. parvum were both associated with urethral inflammation in NCNGU. While a better understanding of the role of U. parvum in heterosexual couples is still needed, it is clear routine testing for M. genitalium should be available for men with NCNGU.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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