Midstream urine culture (MSU) remains the gold standard diagnostic test for confirming urinary tract infection (UTI). We previously showed that patients with chronic lower urinary tract symptoms (LUTS) below the diagnostic cut-off on MSU culture may still harbour bacterial infection, and that their antibiotic treatment was associated with symptom resolution. Here, we evaluated the results of the UK's MSU culture in symptomatic patients and controls. Next, we compared the bacterial enrichment capabilities of the MSU culture with a 50 µl uncentrifuged culture, a 30 ml centrifuged sediment culture, and 16S rRNA gene sequencing. This study was conducted on urine specimens from 33 LUTS patients attending their first clinical appointment (mean age = 49 years, standard deviation [SD] = 16.5), 30 LUTS patients on treatment (mean age = 47.8 years, SD = 16.8) whose symptoms had relapsed, and 29 asymptomatic controls (mean age = 40.7 years, SD = 15.7). We showed that the routine MSU culture, adopting the UK interpretation criteria tailored to acute UTI, failed to detect a variety of bacterial species, including recognised uropathogens. Moreover, the diagnostic MSU culture was unable to discriminate between patients and controls. In contrast, genomic analysis of urine enriched by centrifugation discriminated between the groups, generating a more accurate understanding of species richness. In conclusion, the UK's MSU protocol misses a significant proportion of bacteria, which include recognised uropathogens, and may be unsuitable for excluding UTI in patients with LUTS
