Skip to main content
  • Original Research
  • Open access
  • Published:

Detection of urogenital pathogens in sterile pyuria samples by polymerase chain reaction



Patients with sterile pyuria may be infected with sexually transmitted diseases or have renal tuberculosis. This study investigated the possibility of detecting sexually transmitted diseases and Mycobacterial tuberculosis in sterile pyuria samples with polymerase chain reaction.


Forty-four day-3 negative urine culture samples were investigated for the presence of sexually transmitted diseases and Mycobacterial tuberculosis.


Among the 44 samples, 61.4% were positive by polymerase chain reaction (PCR) for bacterial DNA (either sexually transmitted diseases or Mycobacteria). Among the 27 positive samples, 37% were positive for Ureaplasma urealyticum, 26% were positive for Chlamydia trachomatis, 14.8% were positive for Neisseria gonorrhoeae, 11.1% were positive for Mycoplasma genitalium, 7.4% were positive for Mycoplasma hominis, and only one sample (3.7%) was positive for Mycobacterial tuberculosis. No significant associations were found between PCR-positive urine samples and patient characteristics.


It was concluded that Ureaplasma urealyticum was predominant in sterile pyuria followed by Chlamydia trachomatis. There were no significant associations between PCR-positive samples and sex, symptomatic patients, or antibiotic use. PCR is an instant diagnostic tool for sexually transmitted diseases in sterile pyuria; hence, it is advised to be performed on negative culture samples as a routine laboratory screening test whenever possible.

1 Background

Pyuria is the presence of three or more pus cells per high-power field of centrifuged urine, 10 or more pus cells per cubic millimetre in a urine specimen, or a positive leukocyte esterase urine dipstick test [1]. The presence of more than 5–8 leucocytes per high-power field on microscopy is classified as sterile pyuria (SP) in the absence of positive urine cultures. The diagnosis of SP can be complicated by the presence of casts, proteinuria, and haematuria. It can generally be categorized as either infectious or noninfectious. Noninfectious SP could be caused by radiotherapy, pelvic inflammation, appendicitis, urinary stones, tumours, or physiological changes (pregnancy) [2]. Infectious SP is identified by aerobic laboratory procedures (on a 5% sheep-blood agar plate and MacConkey agar plate) as the prolonged presence of white cells in the urine in the absence of bacteria. Population-based studies suggest that 13.9% of women and 2.6% of men have sterile pyuria, a very common disorder [3]. Historically, colony counts larger than 100,000 colony-forming units (CFU) per millilitre of void urine have been used to distinguish between colonization and bacterial urinary tract infection [3].

Sexually transmitted diseases (STDs) are one of the main causes of SP, and 500 million people worldwide have been reported to be infected with STDs such as gonorrhoea, chlamydia, syphilis, mycoplasma, trichomoniasis, or sexually transmitted viruses [4]. Additionally, SP has been linked to widespread viruses such as adenoviruses and parasitic illnesses such as schistosomiasis [5]. The majority of STDs in men result in symptomatic urethritis, with epididymitis occurring less frequently. Many women may initially experience no symptoms, and pelvic inflammatory disease can progress symptomlessly.

Leukocyte esterase-detecting urine tests in asymptomatic men have a sensitivity of 66.7% for gonorrhoea and 60.0% for chlamydia. Chlamydia trachomatis is the most typical bacterium found in cultures from sexually active populations presenting with sterile pyuria [6]. C. trachomatis and Neisseria gonorrhoeae can be quickly identified using commercially available nucleic acid techniques [7]. Mycoplasma hominis and Ureaplasma urealyticum are among the diseases associated with SP [8].

Atypical infections, in particular renal tuberculosis, should also be taken into consideration in individuals with chronic SP [9]. Patients with chronic sterile pyuria may develop the condition from an unusual infection, such as kidney tuberculosis, that causes the disorder. After lymphadenopathy, genitourinary tuberculosis is the most prevalent type of nonpulmonary tuberculosis, accounting for 27% of cases.

When SP is found, doctors may take a medical history and request laboratory tests, including urine tests for microscopy (fungi, Schistosoma ova), STDs, and Mycobacterial tuberculosis infection [10]. To avoid using unnecessary antibiotics and to encourage antibiotic stewardship, early identification of the cause of SP is crucial; hence, a quick and affordable method for identifying pathogens is necessary, such as polymerase chain reaction (PCR) [11]. According to numerous studies, multiplex PCR compares highly with conventional urine culture in terms of accuracy. Significant time savings are also achieved with multiplex PCR relative to traditional urine culture. This study investigated the presence of STDs and M. tuberculosis in sterile pyuria samples with PCR.

2 Methods

2.1 Study setting

A descriptive cross-sectional study was conducted at a tertiary hospital (in Makkah) in 2022.

2.2 Study design

This is a laboratory-based study.

2.3 Inclusion criteria

Urine samples showing pus cells (10 in the mid-stream urine per cubic millimetre in a urine specimen, or 3 or more white cells per high-power field of unspun urine) and no bacterial growth after 3 days of incubation.

2.4 Exclusion criteria

Urine samples showing bacterial growth within 3 days of incubation.

2.5 Sample size and sampling type

Only urine specimens were used in this study. The Yamane formula used for determining the necessary sample size is as follows: n = N(1 + Ne2), where n = sample size (= 44), N = population size (50 based on the total visits of the patients), and e (significance) = 0.05.

2.6 Patients and clinical samples

In this study, 44 clinical urine samples obtained in 2022 from 44 patients in a tertiary hospital (in Makkah) were analysed. The samples were selected from among those with culture negativity according to standard microbiological methods used in the microbiology laboratory. Only those samples showing no bacterial growth after 3 days of incubation were studied for STDs and M. tuberculosis with PCR. Ethical approval (No. HAPO-02-K-012-2022-09-1171) was obtained from the biomedical research ethics committee at the Faculty of Medicine-Umm Al-Qura University.

2.7 DNA extraction and PCR

Ten millilitres of urine was centrifuged at 10,000 × g for 5 min. Total DNA was then extracted from the pellet using a conventional boiling method. In brief, after being resuspended in 100 μl of water suitable for molecular biology, the samples were centrifuged at 15,000 × g for 10 min. The pellets were reconstituted in 100 μl of water, boiled in a water bath for 15 min at 95 °C, cooled on ice, centrifuged for 2 min at 15,000 g, and then stored at − 20 °C until analysis [12].

PCR was performed using primers (Table 1) selected to detect U. urealyticum, C. trachomatis, N. gonorrhoeae, M. genitalium, M. hominis, and M. tuberculosis, as shown in Table 1. The reaction mixture used for each standard PCR amplification contained deoxynucleoside triphosphates (dNTPs), internal control, primer, DNA polymerase, and a contaminant-prevention solution (Qiagen, USA). Amplification was performed using an Eppendorf Mastercycler PCR machine (Eppendorf, Germany). The cycling conditions included a 5-min initial denaturation period at 95 °C, 30 cycles of 30-s denaturation at 95 °C, 30-s annealing at the proper annealing temperature (Table 1), and a 45-s extension period at 72 °C followed by a final 10-min extension period. After electrophoresis on a 1.5% agarose gel and ethidium bromide staining, the amplification products were observed and photographed using a UVP BioDoc It Imaging System (Cambridge, UK).

Table 1 Primers used in this study

2.8 Statistical analysis

The statistical analysis was performed using SPSS 25 for Windows. The Chi-square (χ2) test was used to find an association between the presence of organisms and patient characteristics (p value < 0.05).

3 Results

Forty-four day-3 negative urine culture samples were studied. Table 2 shows descriptive statistics of the demographic features of the patients. Thus, 40 (91.0%) of the patients were men, while 4 (9%) were women; 22 (50%) were aged 20–40 and 12 (27.3%) were aged 41–60; 7 (15.9%) were asymptomatic and 37 (84.1%) were symptomatic; 11 (25%) were using antibiotics during the study period, while 33 (75%) were not.

Table 2 Demographic features of the patients

The results showed that 27 (61.4%) urine culture samples were positive by PCR for bacterial DNA. Table 3 shows the distribution of the urogenital pathogens in the samples positive for bacterial DNA. Of the 27 positive samples, 10 (37%) were positive for U. urealyticum, 7 (26%) were positive for C. trachomatis, 4 (14.8%) were positive for N. gonorrhoeae, 3 (11.1%) were positive for M. genitalium, 2 (7.4%) were positive for M. hominis, and 1 (3.7%) was positive for M. tuberculosis (Fig. 1). No significant associations were found between PCR-positive urine culture and sex (P = 0.557), the presence of symptomatic patients (P = 0.551), or antibiotic use (P = 0.371).

Table 3 Detection of urogenital pathogens from sterile pyuria by PCR
Fig. 1
figure 1

The genes amplified from urogenital pathogens on 1.5% agarose gel electrophoresis; Lane M: 100 bp DNA ladder

4 Discussion

The traditional diagnostic tools for detecting urine pathogens are culture, antigen detection, and serology; however, the cultivation of organisms such as U. urealyticum, C. trachomatis, N. gonorrhoeae, M. genitalium, M. hominis, and M. tuberculosis is laborious, time-consuming, and requires specific expertise [19, 20]. Such organisms could be diagnosed by molecular testing, which is more sensitive, specific, rapid, and valuable in diagnosing STDs [21, 22]. The high sensitivity of PCR allows the detection of bacterial DNA even when it is present at extremely low concentrations.

The aim of this study was to detect organisms responsible for STDs and M. tuberculosis in sterile urine samples. In this study, 44 day 3 negative urine culture samples were studied. The results showed that 27 (61.4%) urine samples were positive for STDs by PCR. Of the 27 positive samples, 10 (37%) were positive for U. urealyticum, 7 (26%) were positive for C. trachomatis, 4 (14.8%) were positive for N. gonorrhoeae, 3 (11.1%) were positive for M. genitalium, 2 (7.4%) were positive for M. hominis, and 1 (3.7%) was positive for M. tuberculosis. Statistically, there were no significant associations found (P > 0.05) between PCR-positive urine culture and sex, symptomatic patients, or antibiotic use.

Asymptomatic pyuria is the presence of pus cells (greater than 5–9 white blood cells/high-power field) in a properly collected urine sample of a patient who has no signs or symptoms of a urinary tract infection. It was previously found that asymptomatic pyuria may increase the risk of developing overt UTI [23]. In a study of sterile pyuria among admitted patients, only 18.8% of patients with pyuria had a positive culture [24]. In one study, 46.6% of urogenital samples were found to be positive for STDs using PCR, in which U. urealyticum represented 60.7% [25]. Similar studies have reported a higher rate (more than 40%) for U. urealyticum than for M. hominis (more than 20%) [26, 27]. To our knowledge, this is the first study in our area using PCR as a diagnostic tool for STDs. In contrast, other studies reported C. trachomatis as the most prevalent bacterial STD [21, 28]. Such genital pathogens (mycoplasmas) may be related to a low socioeconomic background, such as poverty, and the use of contraceptive drugs [29]. Gonorrhoea has been reported as a cause of sterile pyuria, according to past and present investigations [3, 30]. In one study, more than 1200 symptomatic men with pyuria were found to be infected with C. trachomatis (31%) and M. genitalium (10%) [31]. Another study showed that U. urealyticum was associated with the presence of symptoms and higher rates of pyuria. It was also reported that 37% of patients had mycobacteria isolated from urine with acid-fast bacilli staining, 46% had positive histopathology (bladder biopsies), and 93% had urinary PCR results for M. tuberculosis [32]. The diagnosis of urinary tuberculosis is usually made very late, since it manifests asymptotically with nonspecific signs; hence, it is very important to use a more rapid and sensitive method, such as PCR, for diagnosis [33]. Gonorrhoea has been reported as a common sexual disease in the USA [34]. Additionally, in the WHO statistics for the European Region, in 2008, there were 3.4 million new cases of N. gonorrhoeae [35], placing gonorrhoea in third place after chlamydiosis and syphilis.

Sterile pyuria may be caused by STDs; hence, young, sexually active patients should undergo culture for organisms such as C. trachomatis, which can cause 10% of cases of sterile pyurias [36]. In addition, the fact that bacterial DNA is found in a clinical sample does not necessarily mean that there is an infection. Bacteria may be detected at various sites as a result of colonization or translocation, especially if sensitive methodologies are used. For the detection and identification of live or dead bacteria in situations of suspected infection in patients receiving antibiotic therapy and in symptomatic patients with a culture-negative microbiological report, PCR has proven beneficial.

5 Conclusions

The present study identified 61.4% of negative urine culture samples as positive for STDs with PCR. U. urealyticum was the most frequent (37%), followed by C. trachomatis (25.9%), N. gonorrhoeae (14.8%), M. genitalium (11.1%), M. hominis (7.4%), and M. tuberculosis (3.7%). There were no significant associations found between PCR-positive urine culture and sex, symptomatic patients, or antibiotic use. PCR is an instant diagnostic tool for STDs; hence, it is advised to be performed on negative culture samples as a routine laboratory screening test whenever possible.

Availability of data and materials

The datasets generated in the current study are available from the corresponding author on reasonable request.


C. trachomatis :

Chlamydia trachomatis


Colony-forming units


Deoxynucleoside triphosphate

M. tuberculosis :

Mycobacterial tuberculosis

M. hominis :

Mycoplasma hominis

N. gonorrhoeae :

Neisseria gonorrhoeae


Polymerase chain reaction


Sexually transmitted diseases


Sterile pyuria

U. urealyticum :

Ureaplasma urealyticum


  1. Horan TC, Andrus M, Dudeck MA (2008) CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control 36:309–332.

    Article  Google Scholar 

  2. Bendig DW (2021) The differential diagnosis of sterile pyuria in pediatric patients: a review. Global Pediatric Health 6(8):2333794X21993712.

    Article  Google Scholar 

  3. Longo DL (2015) Sterile pyuria. N Engl J Med 372:1048–1054.

    Article  Google Scholar 

  4. Gottlieb SL, Low N, Newman LM, Bolan G, Kamb M, Broutet N (2014) Toward global prevention of sexually transmitted infections (STIs): the need for STI vaccines. Vaccine 32:1527–1535.

    Article  Google Scholar 

  5. Glen P, Prashar A, Hawary A (2016) Sterile pyuria: a practical management guide. Br J Gen Pract 66(644):e225–e227.

    Article  Google Scholar 

  6. Malhotra M, Sood S, Mukherjee A, Muralidhar S, Bala M (2013) Genital Chlamydia trachomatis: an update. Indian J Med Res 138(3):303–316

    Google Scholar 

  7. Johnson RE, Newhall WJ, Papp JR, Knapp JS, Black CM, Gift TL, Steece R, Markowitz LE, Devine OJ, Walsh CM, Wang S, Gunter DC, Irwin KL, DeLisle S, Berman SM (2002) Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections–2002. MMWR Recomm Rep 18(51(RR–15)):1–38

    Google Scholar 

  8. Nassar, FA, Abu-Elamreen, FH, Shubair, ME, Sharif, FA (2008) Detection of chlamydia trachomatis and mycoplasma hominis, genitalium and ureaplasma urealyticum by polymerase chain reaction in sterile pyuria 53:80–86.

  9. Gibson MS, Puckett ML, Shelly ME (2004) Renal tuberculosis. Radiographics 24(1):251–256.

    Article  Google Scholar 

  10. Vitale AM, Lockwood GM (2020) Urine Microscopy: The Burning Truth – White Blood Cells in the Urine. In: Sharp V, Antes L, Sanders M, Lockwood G (eds) Urine Tests. Springer, Cham.

    Chapter  Google Scholar 

  11. van der Zee A, Roorda L, Bosman G, Ossewaarde JM (2016) Molecular diagnosis of urinary tract infections by semi-quantitative detection of uropathogens in a routine clinical hospital setting. PLoS ONE 11(3):e0150755.

    Article  Google Scholar 

  12. Ahmed OB, Dablool AS (2017) Quality improvement of the DNA extracted by boiling method in gram negative bacteria. Int J Bioassays 6:5347–5349.

    Article  Google Scholar 

  13. Taheri Beni B, Jenab A, Roghanian R, Motamedi H, Golbang N, Golbang P, Zaeimi Yazdi J (2012) Genotyping of endocervical chlamydia trachomatis strains and detection of serological markers of acute and chronic inflammation in their host. Int J Fertil Steril 6(2):101–106

    Google Scholar 

  14. Mahony JB, Luinstra KE, Tyndall M, Sellors JW, Krepel J, Chernesky M (1995) Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens. J Clin Microbiol 33(11):3049–3053.

    Article  Google Scholar 

  15. Zeighami H, Peerayeh SN, Yazdi RS, Sorouri R (2009) Prevalence of Ureaplasma urealyticum and Ureaplasma parvum in semen of infertile and healthy men. Int J STD AIDS 20(6):387–390.

    Article  Google Scholar 

  16. Munoz JL, Goje OJ (2016) Mycoplasma genitalium: An Emerging Sexually Transmitted Infection. Scientifica (Cairo) 2016:7537318.

    Article  Google Scholar 

  17. Blanchard A, Yanez A, Dybvig K, Watson HL, Griffiths G, Cassell H (1993) Evaluation of interspecies genetic variation within the 16SrRNA gene of Mycoplasma hominis and detection by polymerase chain reaction. J Ckin Microbil 31(5):1358–1361.

    Article  Google Scholar 

  18. Eisenach KD, Donald Cave M, Bates JH, Crawford JT (1990) Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis 161(5):977–981.

    Article  Google Scholar 

  19. Lin Y, Fan Y, Cheng C, Sue Y, Hsu Y, Hou C et al (2008) The case | Sterile pyuria and an abnormal abdominal film. ‘Autonephrectomy’ of right kidney. Kidney Int 73:131–133.

    Article  Google Scholar 

  20. Liu T, Lai SY, Zhou W, Liu YL, Chen SS, Jiang YM (2022) Analysis of Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma genitalium and Neisseria gonorrhoeae infections among obstetrics and gynecological outpatients in southwest China: a retrospective study. BMC Infect Dis 22(1):283.

    Article  Google Scholar 

  21. Krõlov K, Frolova J, Tudoran O, Suhorutsenko J, Lehto T, Sibul H, Mäger I, Laanpere M, Tulp I, Langel Ü (2014) Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples. J Mol Diagn 16(1):127–135.

    Article  Google Scholar 

  22. Gdoura R, Kchaou W, Ammar-Keskes L, Chakroun N, Sellemi A, Znazen A, Rebai T, Hammami A (2008) Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples. J Androl 29(2):198–206.

    Article  Google Scholar 

  23. Hwang JH, Park HC, Jeong JC, Ha Baek S, Han MY, Bang K, Cho JY, Yu SH, Yang J, Oh KH, Hwang YH, Ahn C (2013) Chronic asymptomatic pyuria precedes overt urinary tract infection and deterioration of renal function in autosomal dominant polycystic kidney disease. BMC Nephrol 7(14):1.

    Article  Google Scholar 

  24. Hooker JB, Mold JW, Kumar S (2014) Sterile pyuria in patients admitted to the hospital with infections outside of the urinary tract. J Am Board Fam Med 27(1):97–103.

    Article  Google Scholar 

  25. Muhammid HA, Naher HS, Al-Hamadani AH (2015) Detection of Mycoplasma hominis and Ureaplasma Urealyticum in urethritis from sexual active men by real-time PCR. Al-Qadisiah Med J 11(2):60–66

    Google Scholar 

  26. Stellrecht KA, Woron AM, Mishrik NG, Venezia RA (2004) Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas. J Clin Microbiol 42(4):1528–1533.

    Article  Google Scholar 

  27. Domingues D, Tavora Tavira L, Duarte A, Sanca A, Prieto E, Exposto F (2003) Genital mycoplasmas in women attending a family planning clinic in Guine-Bissau and their susceptibility to antimicrobiol agents. Acta Trop 86:19–24.

    Article  Google Scholar 

  28. Abusarah EA, Awwad ZM, Charvalos E, Shehabi AA (2013) Molecular detection of potential sexually transmitted pathogens in semen and urine specimens of infertile and fertile males. Diagn Microbiol Infect Dis 77:283–286.

    Article  Google Scholar 

  29. Peerayeh SN, Sattari M (2006) Detection of Ureaplasma urealyticum and Mycoplasma hominis in endocervical specimens from infertile women by polymerase chain reaction. Middle East Fertility Soc J 11(2):108–108

    Google Scholar 

  30. Rahman MS, Beever W, Skov S, Boffa J (2014) Using urinary leucocyte esterase tests as an indicator of infection with gonorrhoea or chlamydia in asymptomatic males in a primary health care setting. Int J STD AIDS 25:138–144.

    Article  Google Scholar 

  31. Rane VS, Fairley CK, Weerakoon A, Read TH, Fehler G, Chen MY, Bradshaw CS (2014) Characteristics of acute nongonococcal urethritis in men differ by sexual preference. J Clin Microbiol 52(8):2971–2976.

    Article  Google Scholar 

  32. Hemal AK, Gupta NP, Rajeev TP, Kumar R, Dar L, Seth P (2000) Polymerase chain reaction in clinically suspected genitourinary tuberculosis: comparison with intravenous urography, bladder biopsy, and urine acid fast bacilli culture. Urology 56:570–574.

    Article  Google Scholar 

  33. Yazdani M, Shahidi S, Shirani Shirani M (2008) Urinary polymerase chain reaction for diagnosis of urogenital tuberculosis. Urol J 5(1):46–49

    Google Scholar 

  34. Tuddenham S, Hamill MM, Ghanem KG (2022) Diagnosis and treatment of sexually transmitted infections: a review. JAMA 327(2):161–172.

    Article  Google Scholar 

  35. Rowley RTaN F (2012) Global incidence and prevalence of selected curable sexually transmitted infections-2008. In: World Health Organization (ed) World Health Organization. pp 1–20

  36. Goonewardene S, Persad R (2015) Sterile pyuria: a forgotten entity. Ther Adv Urol 7(5):295–298.

    Article  Google Scholar 

Download references


The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (18-MED-1-01-0027).

Author information

Authors and Affiliations



OA helped in formulation of hypothesis, conceptualization, methodology, data collection, writing—original draft and supervision. FB contributed to data collection, writing—review, editing and resources, manuscript revision. Both authors have read and approved the manuscript.

Corresponding author

Correspondence to Omar B. Ahmed.

Ethics declarations

Ethics approval and Consent to participate

The study was approved by the biomedical research ethics committee at Faculty of Medicine-Umm Al-Qura University (Approval No. (HAPO-02-K-012–2022-09–1171). An informed written consent to participate in the study was provided by all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ahmed, O.B., Bahwerth, F.S. Detection of urogenital pathogens in sterile pyuria samples by polymerase chain reaction. Afr J Urol 29, 1 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: