Pooled Antibiotic Susceptibility Testing for Polymicrobial UTI Performs Within CLSI Standards for Validation When Measured Against Broth Microdilution and Disk Diffusion Antibiotic Susceptibility Testing of Cultured Isolates

Background/Objectives: Urinary tract infections (UTIs) by antimicrobial-resistant organisms pose an increasing risk, and clinicians rely on antibiotic susceptibility testing (AST) in their prescribing decisions for these infections. Since standard AST methods performed on isolates, including disk diffusion (DD) and broth microdilution (BMD), have a significantly limited ability to account for interactions between species in polymicrobial infections, we aimed to evaluate the performance of Pooled Antibiotic Susceptibility Testing (P-AST) performed on pellets against isolate AST in polymicrobial clinical urine specimens. Methods: This study is a US-based analysis of consecutive fresh clinical urine specimens submitted for UTI diagnostic testing. Specimens were divided into two analysis groups with either 1) one non-fastidious microorganism or 2) ≥ two non-fastidious microorganisms, ± any number of fastidious microorganisms. Performance characteristics, including essential agreement (EA%), very major errors (VMEs), major errors (MEs), minor errors (mEs), and categorical agreement (CA%), were compared to CLSI standards. Additional analysis to confirm heteroresistance was performed for specimens with P-AST-resistant and BMD/DD consensus-susceptible results. Results: Using Clinical Laboratory Standards Institute (CLSI) standard validation protocols, even without correcting for heteroresistance, we found that the essential agreement between P-AST and standard isolate AST, which compares MICs between P-AST and the “gold-standard” BMD reference method, was ≥ 90%, VMEs were < 1.5%, and MEs were < 3.0%, meeting the CLSI guidelines for AST verification and validation studies. When heteroresistance was accounted for, the P-AST assay performed even better, with VMEs and MEs both < 2.0%. Conclusions: P-AST, a proprietary component of the Guidance® UTI assay (Pathnostics, Irvine, CA), which rapidly measures the susceptibility of a bacterial pellet of cultivable organisms from a urine specimen using a resazurin fluorescent growth probe, performed within FDA and CLSI standards for AST.