Genotype–Phenotype Concordance and Ct-Informed Predictive Rules for Antimicrobial Resistance in Adult Patients with Complicated Urinary Tract Infections: Clinical and Stewardship Implications from the NCT06996301 Trial

<b>Background:</b> Rapid molecular detection of antimicrobial resistance (AMR) can shorten time to effective therapy in complicated urinary tract infections (cUTI), but the ability of gene presence and quantitative PCR signal (Ct, and ΔCt = Ct_marker - IC_Ct) to predict phenotypic non-susceptibility and clinical outcomes requires rigorous evaluation. We analyzed marker-level concordance, Ct→MIC relationships, and the clinical impact pathway in the randomized NCT06996301 trial. <b>Methods:</b> Marker-phenotype concordance metrics (sensitivity, specificity, PPV, NPV, LR+, LR-, κ) were computed for selected marker × species strata with stable sample sizes. Mixed-effects models (log₂[MIC] ~ ΔCt_marker + IC_Ct + collection_method + prior_abx + (1|site)) assessed quantitative Ct→MIC associations. ROC analyses evaluated ΔCt discrimination of phenotypic non-susceptibility. A pre-specified sensitivity analysis included smaller strata (<i>n</i> ≤ 20) with bootstrap 95% confidence intervals for ΔCt slopes and AUCs. Clinical analyses compared PCR-guided (<i>n</i> = 193) versus culture-guided (<i>n</i> = 169) arms for time-to-antibiotic and treatment success using adjusted logistic regression and causal mediation (time-to-antibiotic as mediator; bootstrap inference). <b>Results:</b> High genotype-phenotype concordance was observed for canonical markers (e.g., <i>bla</i>_CTX-M in <i>E. coli</i>: sensitivity 0.94 [95% CI 0.88-0.97], specificity 0.995 [95% CI 0.990-0.998], κ ≈ 0.93). Mixed models showed modest but significant Ct→MIC associations for select markers (e.g., <i>bla</i>_CTX-M in <i>E. coli</i>: ΔCt slope -0.15 [95% CI -0.27 to -0.02], <i>p</i> = 0.015). The sensitivity analysis (<i>n</i> ≤ 20 strata) confirmed consistent negative directions, with robust bootstrap CIs excluding zero for <i>qnrS</i> (<i>E. coli</i>), <i>tetM</i> (<i>E. coli</i>), <i>blaNDM</i> (<i>Klebsiella</i>), and <i>qnrS</i> (<i>Proteus</i>). ROC AUCs for ΔCt prediction of non-susceptibility ranged from 0.62 to 0.81 (95% CIs ≈ 0.47-0.97). Clinically, PCR guidance shortened median time to antibiotic initiation (20 h vs. 52 h) and increased treatment success (88.1% vs. 78.1%; adjusted OR 1.95 [95% CI 1.12-3.40], <i>p</i> = 0.018). Mediation analysis estimated that 63% (ACME 0.112 [95% CI 0.045-0.178], <i>p</i> = 0.002) of the treatment success benefit was mediated through earlier antibiotic initiation. <b>Conclusions:</b> Binary detection of high-impact AMR genes by multiplex PCR reliably predicts phenotypic non-susceptibility and accelerates effective therapy when integrated with stewardship workflows. Quantitative PCR (ΔCt) provides modest but reproducible information about MIC magnitude and may flag heteroresistant subpopulations. A pragmatic implementation model combining rapid PCR with conventional culture is recommended to optimize clinical benefit while retaining isolate recovery for definitive AST.