Abstract Introduction Traditional cultures are used to diagnose penile prosthesis (PP) infections and guide antibiotic therapy. However, false negative results are common, due to biased growth media, non-cultivable organisms, antibiotic usage, or insufficient sample volumes. Next generation sequencing (NGS) circumvents these issues by sequencing DNA directly from the samples. NGS allows for more sensitive and comprehensive identification of microorganisms, which in turn can guide antimicrobial treatment and surgical decision-making. Objective Our goal is to evaluate the performance of NGS-based diagnostics compared to traditional culture in detecting bacteria on infected PPs. Our hypothesis is that NGS will be more sensitive and identify more isolates than traditional cultures. Methods 322 PPs removed for any reason from 2015 to July 2025 were included in the study. During revision surgery, two swab specimens were collected from the pump space if an inflatable PP / corporal space if rod: 1) sent for traditional culture testing at the institutional laboratory, and 2) for NGS testing (MicroGenDx, Lubbock, TX, USA), including targeted 16s rRNA and ITS gene sequencing. Positivity rates, numbers of species, and organism profiles per test were compared. Results NGS had a significantly higher rate of positive detections (n = 111, 34.47%) compared to culture results (n = 40, 12.42%, p0.001). NGS identified significantly more bacterial species than culture (NGS mean = 1.35 (2.55), Culture mean = 0.30 (0.65), p0.001). NGS found 3 fungal species and 55 bacterial species in comparison to the 1 fungal species and 16 bacterial species identified by traditional culture (Figure 1). Unique species found by NGS included many fastidious organisms likely to be missed by culture including various anaerobes, Mycoplasma, and Ureaplasma. Conclusions NGS offers a more sensitive and comprehensive characterization of the organisms contributing to PP microbiome, which may be a useful aid in diagnosis and treatment. Disclosure Yes, this is sponsored by industry/sponsor: Microgen DX Clarification: Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast, Boston Scientific, Focal One, Signati, Microgen DX, Irrimax, Blue Wind, Pfizer
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