B-225 Bridging the Gap Between Clinical Microbiology and Molecular Diagnostics: Correlation of PCR Cycle Threshold Values for Establishing Semi-Quantitative CFU/mL Ranges of Bacterial Targets

Abstract

Abstract Background Molecular diagnostics (MolDx) has surpassed culture in the clinical microbiology laboratory. Wong (2023) states PCR is more sensitive than urine culture in detecting polymicrobial infections (para. 8), marking an advantage in MolDX for urinary tract infections. This advancement poses a challenge for clinicians in treatment decision-making. To address requests for numerical MolDx result ranges, a validation accessed semi-quantitative Colony Forming Units per milliliter (CFU/mL) through the assessment of PCR cycle threshold (CT) values. Methods Bacterial targets of UTI and STI PCR panels were inoculated in nutrient broth (Hardy diagnostics cat#K43) and incubated overnight in a shaking incubator at 35±2°C. The density of the cultures was measured using the OD600 function of a NanoDrop One/One after 18-24 hour incubation. The density of stock cultures was adjusted to 106 CFU/mL. Serial dilutions of stock were made in 1:10 steps covering the range of 106 to 100 CFU/mL. The stock and dilutions were extracted, and PCR was performed in a minimum of 2 replicates of each sample for the UTI and STI PCR panels. Mean CT values, standard deviations and coefficients of variation were calculated for each sample. The linearity of CT values within each organism’s stock and dilution samples were assessed by evaluating the CT differences between each dilution step. Corresponding CFU/mL concentrations were tabulated against each mean CT value. Results The average CT values for reportable CFU/mL ranges were established for each stock and dilution. Mean CT values for each CFU/mL breakdown across all organisms were also calculated, along with standard deviations, to investigate universal CT-CFU/mL correlations. A final interpretation table (Table 1) was constructed according to the universal range calculations. Conclusions The study confirmed correlation between CT values and CFU/mL ranges. Further data analysis and comparison with other laboratories’ results will enhance accuracy and precision of reportable CFU/mL ranges.