A-286 Correlation of PCR and Culture Values in Evaluating respiratory tract infections results: A molecular diagnostic lab approach to microbial detection
Authors: Singh V , Upadhyay P , Vallabhneni A , Alexander B 
Published: October 2024

Abstract

Background
Respiratory tract infections (RTIs) are a major global healthcare burden and a leading cause of morbidity and mortality. While traditional culture remains the “gold standard” for RTI pathogen detection; however, culture results may take up to 48-96 hours rendering providers to treat patients empirically. Advances in PCR-based diagnostic testing have dramatically improved the rapidity and accuracy of RTI detection in patient specimens. Accurate and timely diagnosis of RTIs is not only crucial for targeted treatment and patient management but also towards antimicrobial stewardship. However, the main challenge in adoption of PCR based testing still is the interpretation of PCR results with respect to culture results. In this study, we compared and correlated colony forming unit per milliliters (CFU/mL) from known culture samples to the cycle threshold (Ct) values obtained from multiplex PCR assay to provide accurate and actionable results to healthcare providers.

Methods
Serial dilutions, in sputum, ranging from 10^8 to 10^3 cfu/ml were performed on seven well-characterized RTI-causing Gram-positive and Gram-negative bacterial strains. For each dilution, multiplex PCR testing was performed, while concurrently performing colony counts on solid media. Based on these results, semi-quantitative criteria were derived to compare PCR Ct values to the CFU/ml results obtained from culture results. Quantitative RT-PCR was performed using the nanofluidic OpenArray platform (ThermoFisher, California, USA). All statistical analysis was performed using R version 3.6.0 (R Foundation for Statistical Computing).

Results
This study demonstrated a correlation between the Ct values obtained via PCR and the CFU/ml values obtained from the respiratory culture method. At 10^5 CFU/ml, average Ct of 19.9 and range of 17.67-21.7 (± 1.1 SD) for gram negative bacteria. For the gram positive bacteria, Streptococcus pneumoniae and Staphylococcus aureus, the average Ct value was 23.9, range 19.8-28. (SD +/-4.1). At the clinically relevant 10^5 CFU/ml dilution, gram negative bacteria demonstrated an average Ct value of 23.4, Ct range 22-24.78 (+/-1.64) and the gram positive bacteria displayed an average Ct of 26.79, Range 24.8-28.8, (+/- 2). Based on our studies, we set a Ct value of &lt;22 for gram negative bacteria and &lt;23 for gram positive bacteria to correlate to 2+ growth. We applied the Ct value ranges derived from qRT-PCR to establish a clinically relevant threshold of &gt; and &lt; 10^5cfu/ml, in addition to the traditional interpretation of bacterial growth (1+ - 4+).

Conclusions
This study establishes a direct correlation between qRT-PCR and traditional respiratory microbial cultures. Our results demonstrates the feasibility of quantitative interpretation of molecular results thus assisting providers in making treatment decisions based on PCR results. Given the faster turnaround times and superior sensitivity and specificity of PCR in comparison to microbial culture, our findings provide cogent evidence in favor of PCR based testing for respiratory pathogens for timely and relevant patient care decisions.