The Quantitative Detection of Urogenital Mycoplasmas in Men with Urolithiasis

TL;DR

This study investigates the presence of urogenital mycoplasmas in men with urinary stones and finds higher prevalence in affected individuals compared to healthy controls.

Contribution

The study introduces quantitative real-time PCR to estimate the prevalence of specific urogenital mycoplasmas in men with urolithiasis.

Findings

Urogenital mycoplasma DNA was detected more frequently in men with urolithiasis (43.0%) than in healthy controls (26.6%).

U. parvum was the most commonly detected species, with higher frequency in the study group (38.0%) compared to controls (23.3%).

Abstract

Urease-positive urogenital mycoplasmas are considered to be responsible for the formation of urinary stones. They are usually a part of the normal flora in the human urogenital tract, causing asymptomatic infections. However, many symptomatic infections with these bacteria have been reported. M. genitalium is recognized as a cause of male urethritis and other common genitourinary diseases. The role of other urogenital mycoplasmas is still unclear. The aim of this study was to estimate the quantitative prevalence of Ureaplasma spp., M. genitalium and M. hominis in men with urolithiasis using quantitative real-time PCR (qPCR). The study group comprised 100 men with urolithiasis. A total of 60 men were included in the control group. Urogenital mycoplasma DNA in urine samples was detected significantly more often among men with urolithiasis than in healthy subjects—43.0% vs. 26.6%, p = 0.0382, respectively. The majority of positive results (38/43) concerned U. parvum species, the frequency of which was higher in the study group (38.0% (38/100)) than in the control group (23.3% (14/60)), p = 0.0552. The median concentration of U. urealyticum DNA was higher in the study group compared with the control, p = 0.5714. However, further studies are needed to confirm the usefulness of quantitative studies in determining the role of urogenital mycoplasmas in pathology.

Full text

1. Introduction

Urolithiasis is an important and common health problem in the human population worldwide [1,2]. According to current data, urolithiasis affects both women and men on a comparable scale [3]. Infection with bacteria that synthesize urease may lead to the formation of infection stones by breaking down urea into ammonia and carbonate. An increase in alkaline pH and ammonia is associated with the formation of struvite stones, which are composed of magnesium ammonium phosphate. The most important urease-producing bacteria causing urinary tract infections are Proteus, Providencia and Morganella [4,5]. Ureaplasma spp. strains also produce urease. However, routine urine culture procedures do not include fastidious and slow-growing bacteria [5]. Urogenital mycoplasmas have a tendency to colonize the mucous membranes of the human urogenital tract as commensal inhabitants [6]. These bacteria are associated with NGU (Nongonococcal Urethritis), NCNGU (Nonchlamydial Nongonococcal Urethritis) cervicitis, PID (Pelvic Inflammatory Disease), BV (Bacterial Vaginosis), pathological course of pregnancy, premature birth and infections in newborns, although their role is still discussed [7,8,9,10]. M. genitalium is the only mycoplasma with clearly defined pathogenicity [10]. Ureaplasma spp. are the most prevalent mycoplasma species isolated from the urogenital tract [11]. Colonization by mycoplasmas poses a risk of infection for patients with immunodeficiencies. Moreover, reports on opportunistic infections located outside of the urogenital tract caused by M. fermentans, M. pirum and M. penetrans indicate the importance of colonization with these bacteria [9,12,13]. Conventional polymerase chain reaction (PCR) and real-time PCR have been used for the detection and identification of urogenital mycoplasmas, including those that are rarely included in routine diagnostic procedures. However, the quantitative real-time PCR (qPCR) method is worth considering for its high sensitivity in detection and for explaining the importance of these organisms in pathology.

The formation of deposits in the urinary tract is favored by urinary stasis and urinary tract infections caused by urease-producing bacteria. Therefore, the main aim of this study was to assess the prevalence of urogenital mycoplasmas in patients with urolithiasis and in a control group using the quantitative real-time PCR method.

2. Materials and Methods

2.1. Study Participants

This study group comprised 100 men with urolithiasis (median age 57, range 23–81) and 60 healthy men without urinary tract infections as the control group (median age 47, range 21–68), p = 0.0576. Basic laboratory tests, including morphology, urinalysis and urine culture, were performed in all patients. Patients and the control group were not undergoing antibiotic, chemotherapy and antifungal therapy; STIs were not diagnosed; and they had not undergone any medical procedures for at least 4 weeks prior to this study. All study participants gave written consent and were informed on how to properly collect urine samples for testing.

2.2. Pretreatment of Clinical Specimens and DNA Extraction

Samples of morning first void urine (FVU) (5–10 mL) were collected in sterile plastic containers and transported to the laboratory at +4 °C. The FVU samples (approximately 1 mL) were centrifuged at 15,000× g for 30 min. The supernatant was removed and the pellet was re-suspended in 1 mL of Lyse BG buffer. Genomic DNA was extracted from the pretreated specimens using the Gene MATRIX Bacterial and Yeast Genomic DNA Purification Kit (EURx, Gdańsk, Poland), according to the manufacturer’s protocol. The internal control (IC) was added to each sample as a control to monitor the extraction procedure and detect possible reaction inhibitors.

2.3. qPCR Assays

The AmpliSens Florocenosis/Mycoplasma-FRT real-time PCR test (AmpliSens, Prague, Czech Republic) was used to detect U. parvum, U. urealyticum and M. hominis DNA in 160 FVU samples. The quantitative detection of DNA was based on the linear relationship observed when the fluorescent signal begins to increase exponentially (cycle threshold, Ct). For quantitative detection, the DNA of clinical samples was amplified simultaneously with DNA calibrators (samples with a known concentration of target DNA). The results of DNA calibrator amplification were used to construct a calibration curve and calculate the target DNA concentration in the samples. In the AmpliSens test, the concentration of DNA was determined by the number of genome equivalents of microorganism cells per ml of the clinical sample (GE/mL). These obtained values reflect the absolute concentration of microorganisms in the clinical material. The results of amplification were recorded in four different fluorescence channels (Table 1). The test was performed in accordance with the manufacturer’s instructions using the real-time PCR instrument LightCycler 96 (Roche, Basel, Switzerland).

2.4. Qualitative Real-Time PCR

The AmpliSens Mycoplasma genitalium FRT PCR test (AmpliSens, Czech Republic) was used for M. genitalium DNA detection (targeting the gyrB gene) in accordance with the manufacturer’s instructions. PCR was performed using the real-time PCR instrument LightCycler 96 (Roche).

2.5. Conventional PCR

M. fermentans and M. pirum DNA identification was performed by PCR using specific primers (Table 2) and a Taq PCR Core Kit (Qiagen Inc., Hilden, Germany) in Mastercycler thermocycler (Eppendorf AG, Hamburg, Germany), under the following conditions: M. pirum: 94 °C for 2 min, 35 × (94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min), 72 °C for 5 min; M. fermentans: 94 °C for 2 min, 35 × (94 °C for 30 s, 55 °C for 45 s, 72 °C for 50 s), 72 °C for 5 min. Negative samples were checked for the presence of amplification inhibitors by PCR reactions using beta-globin control primers. Amplified products were visualized under UV light after electrophoresis in 2% agarose gel containing ethidium bromide and recorded using the GeneSys image archiving and analysis system (Syngene, Cambridge, UK). Reference strains M. fermentans ATCC 199989D and M. pirum ATCC 25960D were used as positive controls.

2.6. Statistical Analysis

Statistical analysis was performed using Statistica, version 13 (TIBCO Software INC. 2017, https://www.tibco.com/). Differences between groups were analyzed using the chi-square test (χ2) and Fisher’s exact test. The Shapiro–Wilk test was used to assess the normality of distribution. The Mann–Whitney U test was used to compare medians between groups. A significance level of p < 0.05 was considered statistically significant.

3. Results

Urogenital mycoplasma strains were detected significantly more often in the study group than in the control group (43.0% vs. 26.6%, respectively; p = 0.0382) (Table 3). U. parvum DNA was more frequently found in patients with urolithiasis (38/100, 38.0%) than in healthy men (14/60, 23.3%), p = 0.0552. The DNA of U. parvum was detected more often than that of U. urealyticum in study group (38% vs. 5%, respectively; p < 0.0005). U. parvum was also the most frequent of all mycoplasmas in both groups: patients (38/100 vs. 7/100; p < 0.0005) and controls (14/60 vs. 3/60; p = 0.0041). The presence of M. genitalium and M. pirum DNA was not detected in either group.

In all samples with detected urogenital mycoplasma, the concentration of DNA ranged from >10^2^ to 10^4^ GE/mL. Among the study group, urogenital mycoplasma DNA was detected significantly more often in the lower range (>10^2^–10^3^ GE/mL; 29/100) than in higher range (>10^3^–10^4^ GE/mL; 15/100), p = 0.0253. Similarly, in the control group, samples with urogenital mycoplasma DNA in the range >10^2^–10^3^ GE/mL (11/60) were more frequently detected than those in the range >10^3^–10^4^ GE/mL (6/60), p = 0.0381 (Table 4).

In the study group and control group, the median concentration of urogenital mycoplasma DNA showed no statistically significant difference (7.8 × 10^2^ GE/mL and 6.6 × 10^2^ GE/mL, respectively). No significant difference was found in the median U. parvum DNA concentration between the two groups (7.7 × 10^2^ GE/mL in the study group vs. 6.6 × 10^2^ GE/mL in the control group). The concentration of U. urealyticum DNA was higher in the study group (median = 1.1 × 10^3^ GE/mL) compared with the control group (median = 6.7 × 10^2^ GE/mL), p = 0.5714.

The results of urogenital mycoplasma DNA detection in relation to selected blood and urine test parameters in men from the study group were also analyzed (Table 5).

In all samples from patients with a negative urine culture, urogenital mycoplasma DNA was detected significantly more often in patients with leukocyturia compared to those without leukocyturia (29/43 vs. 15/54, respectively; p = 0.0002) (Table 6).

Leukocyturia was more frequently detected in samples with a higher concentration of urogenital mycoplasma DNA (>10^3^–10^4^ GE/mL; 18/43). No significant differences were found in relation to the other parameters.

4. Discussion

Urolithiasis is one of the most common urological diseases, with an increasing tendency [16,17,18]. Ureaplasma species produce urease, an important factor in the formation of urinary tract stones. Struvite stones are a type of urinary and kidney stone that form almost exclusively in patients infected with urease-positive bacteria. The role of Ureaplasma spp. strains in the stone formation process has been demonstrated by many authors [19,20]. In our study, urogenital mycoplasmas were detected by qPCR in 43% of 100 men with urolithiasis. This was significantly more frequent compared to the controls (p = 0.0382). Mobarak and Tharwat showed the presence of Ureaplasma spp. in the mid-stream urine of 26.7% of men with urolithiasis using the culture method [21]. Using the same method, a higher percentage (62.5%) of urogenital mycoplasmas was detected in patients with urolithiasis, as shown by Shafi et al., but in that study, urine was collected from catheters after nephrolithotomy [22]. The group of men most frequently tested for urogenital mycoplasmas were patients with urethritis, especially NGU. In this group of patients, Maeda et al. showed the prevalence of U. urealyticum and U. parvum in 16.3% and 7.8% of men with NGU, respectively. In an even higher percentage of NCNGU patients, U. urealyticum was detected in 18.8% and U. parvum in 8.8% [23]. In our study group, U. parvum was more frequently detected than U. urealyticum (38% vs. 5%, respectively; p < 0.0005), but our study consisted of patients with urolithiasis. Frølund et al. showed U. urealyticum dominance in patients with acute NGU, whereas U. parvum was dominant in patients with chronic NGU and in the control group [24]. In the development of urolithiasis, both species may be important because both produce urease.

In our research, we also included M. genitalium detection, which is the only mycoplasma with a clearly established role in the etiology of urogenital infections [8,25]. In our study, there were no positive results for M. genitalium; however, taking into account the published results of works by other authors, this species mainly occurs in patients with clinical symptoms of urogenital tract infection [8,26]. Yoshida et al. also did not demonstrate the presence of M. genitalium in the FVU of asymptomatic patients [27]. Strains of this species are rare. Screening studies from the United States, UK, Denmark and Australia have confirmed M. genitalium infection in 1–3% of men and women [28,29,30,31]. The frequency of DNA detection for this species increases significantly in sexually transmitted disease clinic patients. Among men treated at the STI clinic in Seattle (USA), the percentage of people testing positive for this pathogen was 9% [32]. In another study from France, in young men with symptoms of urethritis, the presence of M. genitalium DNA was detected in 21.7%. In the same study, in men without urinary tract infections, M. genitalium DNA was not detected [33].

Many authors emphasize that M. fermentans, M. pirum and M. penetrans may be etiological agents of opportunistic infections in at-risk patient groups, especially in immunocompromised individuals. Preiswerk et al. drew attention to the possibility of M. penetrans infections occurring in immunocompromised patients, describing the case of a 38-year-old woman after a kidney transplant who was diagnosed with bacteremia of this etiology [34]. Nevertheless, the occurrence of M. fermentans, M. penetrans and M. pirum species and their pathogenic potential have not been sufficiently examined or confirmed, and the reported cases still mainly concern patients infected with HIV. The presence of M. fermentans was confirmed in the kidney tissue of patients with nephropathy and in the bronchopulmonary lavage of AIDS patients with diagnosed pneumonia who developed respiratory distress syndrome [35,36]. In a different study among women (pregnant and infertile) without pathology, HIV infection or nephropathy, the prevalence of Mycoplasma fermentans was noted in 14.1% of these patients. Furthermore, M. fermentans has been found to be associated with bacterial vaginosis [37]. This may indicate that M. fermentans may be involved in changes to the vaginal microbiome, and research into these rarely detected microorganisms should be continued.

4.1. Quantification of Urogenital Mycoplasmas in Patients with Urolithiasis and in the Control Group

U. urealyticum strains are more often indicated as the cause of symptomatic infections, mainly in the case of NGU in men. In our study, the concentration of U. urealyticum DNA was higher in patients (median = 1.0 × 10^3^ GE/mL) than in the controls (median = 6.7 × 10^2^ GE/mL). The concentration of tested DNA in our study group was similar to the group with acute NGU in the study conducted by Frølund et al. They showed that the bacterial load of U. urealyticum was higher in both acute NGU (median = 6.7 × 10^3^ GE/mL) and chronic NGU (median = 5.5 × 10^4^ GE/mL) than in the control group (median = 313 GE/mL) (p = 0.002 and p = 0.02, respectively). The bacterial load of U. parvum was similar in the NGU groups compared to the control group [24]. The concentration of U. parvum DNA in our study and the control group did not show a statistically significant difference. Strauss et al. showed that a strong positive result (by semi-quantitative PCR test in urine samples) for U. parvum occured significantly more often in symptomatic patients compared to asymptomatic patients. However, these strongly positive samples, examined by qPCR, exceeded 7.5 × 10^5^ GE/mL [38]. U. parvum at lower concentrations of GE/mL were often found in subjects without symptoms of infection. Deguchi et al. reported that the bacterial load of U. parvum in the male urethra was less than 5 × 10^3^ cells of U. parvum/mL in most cases (83%). The authors concluded that U. parvum typically colonizes the male urethra, but in cases with high levels of these strains, an inflammatory response may occur [39].

4.2. The Importance of Leukocyturia Among Urogenital Mycoplasma Infections

Leukocyturia is present in both infectious and non-infectious urinary tract diseases. Wanic-Kossowska et al. pointed out that in the case of leukocyturia and a negative urine culture test, the infection may be caused by Mycoplasma spp. and/or U. urealyticum [40]. In our study, leukocyturia was found significantly more often in patients with a positive test result for urogenital mycoplasmas compared to a negative result. Deguchi et al. showed that the presence of U. parvum DNA at a load of ≥5  ×  10^3^ cells/mL in FVU of the studied men was correlated with the presence of leukocyturia (>12.5 leukocytes/µL in FVU) [38]. In our study, leukocyturia was more often detected in patients with a higher concentration of urogenital mycoplasma DNA (>10^3^–10^4^ GE/mL). Dupin et al. found M. genitalium in 21.7% of symptomatic patients with urethritis and leukocyturia, whereas these bacteria have not been detected in patients without leukocyturia [33]. Vlasic-Matas et al. found that in patients with recurrent urinary tract infections, leukocyturia was more commonly associated with U. urealyticum than with C. trachomatis infection [41]. The study by Park and Lee also showed an association between the prevalence of U. urealyticum and an increased number of leukocytes in urine (>5 WBC) [42]. The results of our study, along with those of previous studies, showed a significant association between leukocyturia and the presence of urogenital mycoplasmas. This confirms that patients with symptoms of urogenital infection and a negative urine culture test (for pathogens in routine urine testing) should be tested for atypical pathogens, such as urogenital mycoplasmas.

5. Conclusions

In our study, we found that urogenital mycoplasma DNA among men with urolithiasis occurred more frequently than in the control group. The concentration of U. urealyticum DNA was higher in the study group compared with the control group; however, further studies are needed to confirm the usefulness of quantitative studies in determining the role of urogenital mycoplasmas in pathology. This study also showed the association between leukocyturia and the presence of urogenital mycoplasmas in the examined patients. Therefore, in cases where determining the etiological agent of urogenital infection is difficult in symptomatic patients with confirmed leukocyturia, diagnostic testing for M. hominis, U. urealyticum and U. parvum should be recommended.