Molecular Tests Reveal Hidden Pathogens in Sterile Pyuria Cases

A prospective cross-sectional study published in the Annals of Clinical Microbiology and Antimicrobials has identified high rates of atypical microbial DNA in patients diagnosed with sterile pyuria. The research, conducted in the microbiology laboratories of two university hospitals in Türkiye, utilized real-time multiplex polymerase chain reaction (PCR) to detect pathogens that conventional urine cultures often miss. The study found that molecular testing detected at least one potential pathogen in 50.2% of patients with sterile pyuria, a rate significantly higher than the 10% observed in a control group. These findings suggest that a substantial portion of patients with sterile pyuria may harbor specific microorganisms that require further clinical correlation, particularly Ureaplasma, Mycoplasma, and Gardnerella vaginalis.

The study aimed to determine the frequency of microbial DNA detection in patients with sterile pyuria and to evaluate the association between leukocyte count, leukocyte esterase test results, and physicians’ treatment decisions. Sterile pyuria is defined as the presence of at least 10 leukocytes per milliliter in uncentrifuged urine and/or a positive leukocyte esterase test on a urine dipstick, despite a negative urine culture. The research team analyzed urine samples from 201 patients meeting these criteria and 180 control patients who had negative urine cultures and no significant pyuria. The sample was drawn from 6,629 urine samples submitted with urinary system infection diagnostic codes between September 1 and November 1, 2024.

Midstream urine samples were collected following appropriate pre-cleaning of the external urethra to minimize contamination. Samples were inoculated onto blood agar and EMB agar media and incubated aerobically for 18 to 24 hours. For molecular analysis, DNA was isolated using the EZ1 Virus Mini Kit v2.0 and the EZ1 Advanced XL automated nucleic acid extraction system. The presence of 14 potential pathogens was investigated using the NeoPlex STI-14 kit on a Rotor-Gene Q MDx 5Plex HRM real-time PCR device. This panel targeted 10 bacteria, two viruses, one yeast, and one protozoon, including Mycoplasma hominis, Ureaplasma urealyticum, and Chlamydia trachomatis.

The results demonstrated a stark contrast between the patient and control groups. In the patient group, at least one possible pathogen was detected in 101 samples, representing 50.2% of the cohort. In the control group, a possible pathogen was identified in only 18 samples, or 10%. The most frequently detected microorganisms in the sterile pyuria patients were Ureaplasma urealyticum, found in 21.9% of cases, Mycoplasma hominis in 12.9%, and Gardnerella vaginalis in 10.9%. In the control group, the most common detections were Ureaplasma urealyticum at 2.2%, Ureaplasma parvum at 1.7%, and Mycoplasma hominis at 1.7%. The study noted that the 10% positivity in controls likely reflects background colonization or low-level contamination rather than active infection.

Statistical analysis using logistic regression revealed significant associations between laboratory findings and PCR positivity. A leukocyte count of at least 10 per milliliter was significantly associated with a positive PCR result, with an odds ratio of 6.84. The presence of both significant leukocyte counts and positive leukocyte esterase results was also strongly linked to PCR positivity. However, the study found that treatment initiation was more frequent in the patient group than in controls, at 43.3% versus 30%. Logistic regression indicated that leukocyte counts of at least 10 per milliliter were significantly associated with physicians’ treatment decisions, with an odds ratio of 2.59, while leukocyte esterase positivity alone was not significantly associated with treatment initiation.

The demographic profile of the participants showed a median age of 56 years, with a female-to-male ratio of 58.5% to 41.5%. The median leukocyte count across all samples was 16 per milliliter. Among the patients with sterile pyuria, the detection of Ureaplasma and Mycoplasma species was particularly prevalent. Approximately two-thirds of the patients had these species as single possible agents, and this proportion rose to about three-quarters when multiple detections were included. The study highlighted that these organisms lack a cell wall, making them invisible to Gram staining and difficult to detect via routine culture, which often leads to their misinterpretation as mere colonization rather than potential pathogens.

Gardnerella vaginalis emerged as another significant finding, detected in 10.9% of the patient group compared to 1.1% in the control group. Interestingly, 27.3% of the patients positive for Gardnerella vaginalis in the patient group were male, indicating that this bacterium is not limited to female genital tract colonization. The study noted that in the patient group, the highest frequency of concurrent significant leukocyte counts and positive leukocyte esterase results occurred in patients with Gardnerella vaginalis, with 95.5% of these cases showing both markers. This supports the potential clinical relevance of this bacterium in symptomatic patients with sterile pyuria.

The study also identified other pathogens, including Chlamydia trachomatis in three patients, two women and one man aged 45 to 72 years. This finding is notable because Chlamydia is traditionally considered uncommon in middle-aged and older adults. Additionally, Streptococcus agalactiae and Candida albicans were detected in small numbers, suggesting low-level presence below the threshold for significant culture growth. The detection of Neisseria gonorrhoeae and HSV-2 in the patient group was consistent with negative culture results, further illustrating the limitations of conventional methods for certain pathogens.

A major limitation of the study is the use of qualitative PCR, which detects the presence of microbial DNA but cannot quantify pathogen load or definitively distinguish between active infection, colonization, or contamination. The 10% PCR positivity rate in the control group underscores this challenge, as it likely reflects urogenital colonization or subclinical carriage. The authors emphasize that until quantitative PCR studies establish pathogen load thresholds, the findings should be interpreted as the identification of candidate pathogens requiring clinical correlation rather than definitive etiologic diagnosis. The study also lacked detailed comorbidity and clinical-context data, which restricted the ability to adjust for potential confounding variables in regression analyses.

The correlation between treatment administration and PCR positivity was found to be low but significant, with a correlation coefficient of 0.33. This suggests that clinicians’ treatment decisions were influenced by additional clinical factors beyond PCR results, which were not available to them at the time of evaluation. The study noted that approximately one-third of the control group, whose results did not support infection, received antibiotics, reinforcing concerns about the appropriate use of antimicrobial drugs. The authors argue that high-evidence-level laboratory findings are increasingly important to ensure appropriate antimicrobial use and to control rising resistance, which the World Health Organization has declared a global emergency.

The study was conducted over a two-month period in a single geographic region, which limits the assessment of seasonal variation and generalizability to other populations. The absence of follow-up clinical outcome data prevented a direct evaluation of the long-term clinical significance of PCR positivity. Despite these limitations, the study provides valuable insights into the microbial etiology of sterile pyuria. It highlights the need for advanced diagnostic technologies to clarify the true status of patients with diagnostic challenges and to improve routine clinical practice.

The findings suggest that molecular testing can identify a significant proportion of patients with sterile pyuria who harbor specific pathogens, particularly Ureaplasma, Mycoplasma, and Gardnerella vaginalis. These organisms are often overlooked in routine diagnostic workflows due to their fastidious growth requirements and the technical limitations of conventional culture. The study underscores the importance of considering these atypical agents in the differential diagnosis of sterile pyuria, especially in adult women with elevated leukocyte counts and positive leukocyte esterase tests. Further research using quantitative methods is needed to establish clinical thresholds for treatment and to better understand the role of these microorganisms in urinary tract health.

Subject of Research: Investigation of atypical microbial agents in patients with sterile pyuria

Article Title: Investigation of atypical microbial agents in patients with sterile pyuria

Article References: Tanrıverdi, E. S., Yakupoğulları, Y., Gündüz, A., & Otlu, B. (2026). Investigation of atypical microbial agents in patients with sterile pyuria. Annals of Clinical Microbiology and Antimicrobials, 25(1), Article 34. https://doi.org/10.1186/s12941-026-00874-3

Image Credits: AI Generated

DOI: 10.1186/s12941-026-00874-3

Keywords: Investigation, atypical, microbial, agents, patients, sterile, pyuria, scientific research