Species-Level Reconfiguration and Retrospective Bridging Assessment of a Multiplex PCR Panel for Complicated and Recurrent Urinary Tract Infections

Background: Complicated and recurrent urinary tract infections (cUTI/rUTI) remain diagnostically challenging, particularly after antimicrobial exposure and in polymicrobial or fastidious infections. Multiplex PCR may address some limitations of culture, but panel composition requires periodic refinement as epidemiological, resistance, and clinical evidence evolves. This study describes the transition of the DocLab UTM™ multiplex PCR assay from the prospectively evaluated V2 configuration to the redesigned V5 configuration. Methods: V2 pathogen-level agreement with quantitative urine culture and molecular AMR target–phenotypic AST agreement were characterized using 773 baseline specimens from the prospective multicenter NCT06996301 study. V2-to-V5 panel reconfiguration incorporated US epidemiological and AMR evidence, clinical and antimicrobial-stewardship considerations, and species-level reporting value. V5 was assessed retrospectively using 743 evaluable archived specimens from the same NCT06996301 cohort. Pathogen and AMR agreement were evaluated against the original parent-study quantitative culture and phenotypic AST comparators, and directly shared V2–V5 targets were compared within the common paired cohort using McNemar testing with Holm adjustment for multiple target-specific comparisons. Six newly introduced pathogen targets and one internal process-control target underwent limited contrived-urine feasibility testing. Results: V2 showed high agreement for most established pathogen and AMR targets, although lower negative percent agreement was observed for selected composite pathogen groups. V5 separated several composite targets into species-level outputs and modified selected AMR reporting, principally the qnr architecture. Across evaluated V5 AMR targets, positive percent agreement ranged from 94.9% to 100.0% and negative percent agreement from 96.6% to 99.6%. After Holm adjustment, paired V2–V5 comparisons identified no statistically significant differences in molecular classifications among the directly shared pathogen or AMR targets (all adjusted p > 0.05). All tested spiked-positive and negative contrived specimens were classified as expected under the predefined feasibility conditions. Conclusions: V5 increased species-level reporting resolution while retaining generally high retrospective pathogen and AMR agreement with the original parent-study comparators. Paired analyses did not identify statistically significant directional changes in shared-target classifications. Newly introduced targets require comprehensive validation, and prospective evaluation of the final V5 configuration is needed before conclusions regarding clinical or stewardship benefit can be made.