Sexually transmitted infections (STIs) remain a major global public health concern, with a high proportion of asymptomatic cases. Undiagnosed infections may lead to significant clinical, economic, and epidemiological consequences. Patients with sterile pyuria and suspected urinary tract infection (UTI) represent a relevant target population for STI screening.
MethodsWe evaluated the real-world performance of a targeted STI screening strategy based on urine sample pooling in patients with suspected UTI, genitourinary symptoms, leukocyturia, and negative urine culture.
ResultsOver a 15-month period, 2019 urine samples were analysed using pooled PCR testing, identifying 212 STI-positive patients (10.5%). Chlamydia trachomatis was the most prevalent pathogen, followed by Trichomonas vaginalis, Mycoplasma genitalium, and Neisseria gonorrhoeae. Pooling did not compromise analytical sensitivity, with comparable cycle threshold values between pooled and individual samples.
ConclusionsPooled PCR screening reduced testing volume while preserving diagnostic yield, supporting its use as an efficient and potentially cost-effective strategy for STI-screening.
Las infecciones de transmisión sexual (ITS) constituyen un problema de salud pública, con una elevada proporción de casos asintomáticos. Los pacientes con piuria estéril y sospecha de infección del tracto urinario (ITU) representan una población diana útil para el cribado de ITS.
MétodosSe evaluó el rendimiento en práctica clínica real de una estrategia dirigida de cribado de ITS basada en el pooling de muestras de orina en pacientes con sospecha de ITU, síntomas genitourinarios, leucocituria y urinocultivo negativo.
ResultadosDurante 15 meses se analizaron 2019 muestras mediante PCR en pools, identificándose 212 pacientes positivos (10,5%). Chlamydia trachomatis fue el patógeno más frecuente, seguido de Trichomonas vaginalis, Mycoplasma genitalium y Neisseria gonorrhoeae. El pooling no comprometió la sensibilidad analítica, (Ct comparables entre muestras agrupadas e individuales).
ConclusionesEl cribado mediante PCR en pools optimiza el rendimiento diagnóstico, constituyendo una estrategia eficiente y potencialmente coste-efectiva para el cribado de ITS.
Sexually transmitted infections (STIs) are a global public health concern due to both their clinical impact and their economic burden on healthcare systems.1,2 Many bacterial STIs are asymptomatic. In women, over 60% of Neisseria gonorrhoeae infections and more than 80% of Chlamydia trachomatis infections produce no symptoms.3 The absence of diagnosis in such cases may lead to serious complications such as tubal disease, infertility, or pelvic inflammatory disease in women, and orchitis or epididymitis in men, with substantial clinical, social, economic, and epidemiological implications.4
Preventive strategies are therefore essential, both in the form of primary prevention—including promotion of prophylactic measures and sexual health education—and secondary prevention, which relies on case detection and appropriate treatment. To improve the detection of new infections, the scientific community highlights the importance of case reporting, opportunistic testing, and systematic screening.5
Previous studies have focused on patients with suspected urinary tract infection (UTI), as clinical presentation often overlaps with STIs. In particular, sterile pyuria in patients with suspected UTI has been associated with the presence of STIs.6–8 This scenario has been linked to UTI overdiagnosis and unnecessary empirical antibiotic therapy, alongside underdiagnosis of STIs.9,10
Between January 2024 and May 2024, we evaluated different STI screening strategies in patients with suspected UTI, considering clinical symptoms, sex, and leukocyturia as the main decision parameters for defining an algorithm that maximised diagnostic yield. We concluded that the most cost-effective approach was screening through pools of four mid-stream urine samples from patients aged 15–55 years, with leukocyturia ≥70leukocytes/μL and a negative, non-significant or contaminated urine culture.11 Based on these findings, this screening strategy was implemented in routine clinical practice in our health department from July 2024 onwards.
The objective of this study is to assess the real-world performance of this strategy following its implementation, with a particular focus on its diagnostic effectiveness in routine clinical practice.
Materials and methodsStudy designWe conducted a retrospective observational study evaluating STI screening in urine samples meeting the predefined inclusion criteria over a 15-month period.
PatientsAll urine samples submitted with suspected cystitis between August 2024 and October 2025 to the Clinical Microbiology Department of Hospital Clínico Universitario de Valencia, Spain, were assessed. Samples were included if they met the following criteria: (1) male or female patients aged 15–55 years; (2) symptoms compatible with UTI; (3) positive urine flow cytometry with ≥70 leukocytes/μL; and (4) negative, non-significant or contaminated urine culture. The current study was approved by the Research Ethics Committee of Hospital Clínico Universitario INCLIVA (N∘2024/314).
Microbiological proceduresUrine culture screening was initially performed using the Sysmex UF-5000 flow cytometer according to the manufacturer's guidelines. Samples were considered positive when ≥40leukocytes/μL and/or ≥140bacteria/μL for men, or ≥240bacteria/μL for women. Positive samples were plated quantitatively on Becton-Dickinson CHROMagar™ Orientation Medium and evaluated according to national microbiology guidelines.12 Negative, non-significant or contaminated samples meeting the eligibility criteria were retrieved for real-time PCR using Abbott Alinity m STI AMP, detecting C. trachomatis (CT), N. gonorrhoeae (NG), Mycoplasma genitalium (MG), and Trichomonas vaginalis (TV), together with an internal PCR control and a cellularity control (β-globin). Urine samples requiring Gram staining (from emergency and inpatient hospital settings) were pooled in groups of four using the sediment after centrifugation at 2500rpm for 10min, combining 200μL from each specimen. Outpatient urine samples were pooled directly from the original urine. If a pool tested positive, individual samples were subsequently analysed. Pools exhibiting PCR inhibition or failure to amplify β-globin were excluded from further analysis. Only in specific circumstances—typically before public holidays and according to laboratory needs—were samples processed in smaller pools than usual.
Statistical analysisDescriptive statistics were performed using XLStat (Microsoft Corporation). Median values were compared using Mann–Whitney U test. Significance was defined as p<0.05. Figures were generated using GraphPad Prism 6.
ResultsWe analysed 2075 urine samples grouped into 534 pools (482 pools of 4 samples, 43 pools of 3 samples, and 9 pools of 2 samples). Fourteen pools, corresponding to 56 urine samples, showed PCR inhibition or absence of β-globin detection and were therefore excluded from the analysis. There were 279 male and 1740 female patients, with a mean age of 34.31 years (SD 11.034). The main indication for urine culture was urinary symptoms (40.06%), followed by routine medical check-ups (25.60%) and abdominal, iliac fossa, lumbar or hypogastric pain (10.4%). A total of 44.3% of samples originated from the Emergency Department (Table 1).
Clinical and demographic features.
| Gender, n (%) | Female | Male |
|---|---|---|
| 1740 (86.2) | 279 (13.8) | |
| Age, median (range) | 33 (15–55) | 39 (15–55) |
| Indication for urine culture, n (%) | ||
| Urinary symptoms | 678 (38.9) | 131 (46.9) |
| Routine medical check-ups | 506 (29.1) | 11 (3.9) |
| Abdominal, iliac fossa, lumbar or hypogastric pain | 194 (11.1) | 16 (5.7) |
| Renal colic | 89 (5.1) | 32 (11.5) |
| Unrelated fever | 33 (1.9) | 3 (1.1) |
| Other | 240 (13.8) | 86 (30.8) |
| Origin of request, n (%) | ||
| Emergency department | 755 (43.4) | 140 (50.2) |
| Hospital ward | 136 (7.8) | 57 (20.4) |
| Outpatient care | 849 (48.8) | 82 (29.4) |
| Urine culture results, n (%) | ||
| Non-significant bacterial count | 557 (32.0) | 66 (23.7) |
| Negative culture | 838 (48.2) | 185 (66.3) |
| Contaminated culture | 345 (19.8) | 28 (10.0) |
| STI PCR results, n (%) | ||
| Positive | 159 (9.1) | 53 (19.9) |
In total, 212 patients (10.5%) were positive for at least one STI, detected across 190 pools. Twenty-two pools contained two positive patients. By decreasing prevalence, CT was the most frequently detected pathogen (n=75, 35.4%), followed by TV (n=48, 22.6%), MG (n=45, 21.2%), and NG (n=22, 10.4%). There were 22 coinfections. CT was involved in 14 coinfections, 6.6% (8 with MG, 4 with NG, 2 with TV). MG coinfections, in addition to those already mentioned with CT, accounted for 7 cases, 3.3% (2 with NG, 5 with TV). There was one coinfection involving TV and NG. No cases involved three or more pathogens. A significantly higher proportion of positive results was observed in men compared with women (19.9% vs 9.1%; p=0.043).
Given the particular nature of urine samples reported as contaminated in culture, this subgroup was analysed separately. Among contaminated urine samples, 31 of 373 patients (8.3%) had a positive STI result.
Cycle threshold (Ct) values were recorded during the first 8 months (August 2024–March 2025). The largest Ct difference between pooled and individual samples was observed for NG (median +2.5), followed by CT (+1). Median Ct values for TV and MG were identical in pooled vs individual samples. None of these differences reached statistical significance (Fig. 1).
Discussion
Active STI screening has long been recognised as a key strategy for early case identification, interruption of transmission chains, and prevention of complications associated with untreated infection.
Selection and evaluation of diagnostic optimisation strategies should therefore be a priority for healthcare systems and microbiology services, as part of Microbiological Diagnostic Stewardship Programmes (PRODIM).
In our previous study, we reported an STI prevalence of 7.5% among patients screened despite no initial suspicion of STI.11 Using our optimised strategy, driven primarily by leukocyturia ≥70/μL, positivity increased to 10.5%, demonstrating improved diagnostic efficiency. Conversely, the positivity rate in men was substantially higher than in women, suggesting that overly restrictive criteria may be being applied in males. Relaxing these selection criteria could optimise the diagnostic strategy by increasing the number of new diagnoses in men.
Pooling four urine samples did not reduce analytical sensitivity: although pathogen load is diluted, molecular testing performance was retained, with no significant Ct differences. While samples near assay detection limit may be missed, we did not observe any case where individual testing detected an infection not already identified in the pooled sample.
Previous studies have reported STI prevalence in women with UTI-like symptoms ranging from 3.4% to 23%,9,13,14 highlighting the importance of pre-test probability and appropriate target population selection. In a Canadian cohort, STI testing increased from 5.5% to 45.2%, indicating greater screening coverage but at the cost of reduced diagnostic efficiency.14
In our series, 534 pools identified 212 STI-positive individuals. Assuming only one infected sample per positive pool, a total of 1382 PCR tests would have been required (534 pooled+848 individual). As we observed 190 positive pools, we ultimately performed 1281 PCRs, representing a saving of 738 assays. It is also important to note that a proportion of patients with undiagnosed STIs may progress to more severe complications, including pelvic inflammatory disease (PID) or tubal damage, potentially requiring hospitalisation. Such outcomes entail additional healthcare expenditure and may substantially impair patient quality of life.
One concern in screening strategies is overtreatment of low-clinical-impact pathogens. Although the test panel detects four established primary pathogens, the role of routine MG screening remains debated, particularly in asymptomatic patients.15 The main limitations of the study include the absence of clinical impact, treatment outcome, and follow-up assessment; the potential exclusion of patients with STIs inherent to the applied selection criteria; and the monocentric design, which may limit generalisability.
In conclusion, STI screening in patients with symptoms of cystitis, leukocyturia, and negative urine culture is an efficient and potentially cost-effective approach for identifying otherwise undetected infections within our setting.
Authors’ contributionsPablo Gil, Katherine Villarruel, Javier Guillem, and Silvia Hernández contributed to validation, methodology, and data curation. David Navarro contributed to study conceptualization and supervision. Eliseo Albert contributed to conceptualization, formal analysis, data curation, and drafting of the original manuscript. All authors reviewed and approved the final version of the manuscript.
Ethical considerationsThe current study was approved by the Research Ethics Committee of Hospital Clínico Universitario INCLIVA (N∘2024/314).
Informed consentDue to the retrospective nature of the study, the ethics committee waived the requirement for informed consent.
Use of artificial intelligenceDuring the preparation of this manuscript, the author(s) used ChatGPT to assist with the writing process. After using this tool/service, the author(s) reviewed and edited the content as needed and take full responsibility for the final version of the publication.”
FundingThe current work received not public or private funds.
Conflict of interestDavid Navarro is part of the Editorial board of Enfermedades Infecciosas y Microbiología Clínica and declare that they have remained outside the evaluation and decision-making process in relation to this article. The rest of the authors declare no conflict of interest.
