Semi-Quantitative ΔCt Thresholds for Bacteriuria and Pre-Analytic Drivers of PCR-Culture Discordance in Complicated UTI: An Analysis of NCT06996301

Background: Quantitative urine culture (CFU/mL) remains the reference standard for diagnosing urinary tract infections (UTIs) but is limited by delayed turnaround times and sensitivity to pre-analytic factors. Multiplex PCR panels offer rapid detection; however, standardized mappings between molecular signals and viable bacterial burdens are insufficiently defined. We used the multicenter NCT06996301 paired dataset to evaluate the analytical validity (AV), clinical validity (CV), and pre-analytic robustness of ΔCt (Ct_target − IC_Ct) as a semi-quantitative indicator of bacterial load. Methods: We analyzed 1027 paired PCR and quantitative urine culture specimens from six sites. The primary molecular predictor was ΔCt (Ct_target − IC_Ct). Species-level Spearman and Pearson correlations, species-specific linear mixed-effects calibration models (log₁₀CFU ~ ΔCt + (1|site)), and ROC analyses were performed for the taxa meeting pre-specified sample thresholds. A pooled multilevel model assessed the collection method and time-to-processing (hours) effects (log₁₀CFU ~ ΔCt × collection_method + ΔCt × time_to_processing_h + (1|site) + (1|run)). AV was assessed via reproducibility, internal control normalization, and site run variance. CV was assessed by ΔCt calibration and discrimination. Clinical utility (CU) was contextualized using outcomes from the parent randomized trial. Results: PCR positivity exceeded culture positivity across all sites (PCR ~82–88% vs. culture ~66–70%); this excess likely reflects a combination of molecular detection of non-viable DNA, detection of fastidious taxa less readily recovered by culture, and pre-analytic effects. For six common uropathogens (n = 90 pairs/species), ΔCt correlated strongly with log₁₀CFU (Spearman ρ = −0.64 to −0.75; Pearson r = −0.75 to −0.83). Species-specific mixed models yielded slopes of −0.746 to −0.922 log₁₀CFU per ΔCt unit (all p < 0.001), indicating that each 1 unit ΔCt change corresponds to a ~5.6–8.4-fold CFU difference. ROC AUCs for ΔCt discrimination were 0.78–0.84 (interpreted as good discrimination, i.e., ΔCt meaningfully improves the clinician’s probability estimate of a high CFU but does not perfectly classify every specimen). The collection method (catheter vs. clean-catch) did not materially modify the ΔCt→CFU relationship, whereas the processing delay was associated with reduced recovered CFU (~0.048 log₁₀CFU lost per hour) and a significant ΔCt × time interaction, consistent with time-dependent viability loss driving the PCR⁺/culture⁻ discordance. Conclusions: ΔCt from the DOC Lab UTM 2.0 panel shows a reproducible, analytically valid semi-quantitative measure of urinary bacterial load. Laboratories can derive assay- and workflow-specific ΔCt cut points for semi-quantitative reporting, but thresholds must be validated prospectively and paired with operational controls for specimen handling.