Full Validation of Pooled Antibiotic Susceptibility Testing Using CLSI Methods and Performance Criteria in UTI Pathogens

Background: Here, we validate a unique susceptibility assay, Pooled Antibiotic Susceptibility Testing (P-AST), used for complicated, persistent, and recurrent urinary tract infections (UTIs), following Clinical and Laboratory Standards Institute (CLSI) protocols and performance metrics. Methods: P-AST™ was validated against the standard disk diffusion method with discrepancy resolution by the broth microdilution (BMD) reference method and error resolution by triplicate repeat testing against BMD. Performance was evaluated for five groups of non-fastidious uropathogenic organisms (Enterobacterales, Enterococci, Staphylococci, Pseudomonas aeruginosa, and Acinetobacter species) for up to 20 antibiotics, as clinically relevant per group. Fresh and frozen clinical urine specimens with non-fastidious bacteria detected, as well as pre-characterized frozen “challenge” isolates, were included. Results: P-AST met CLSI target performance criteria of: ≥ 90.0% categorical agreement, < 3.0% very major error, < 3.0% major error, minor error ≤ 10.0% or within laboratory standards, and precision (reproducibility) > 95.0% across all analysis groups. Across all monomicrobial organism group analyses, there were no very major errors (VMEs) and two major errors (MEs). Across all polymicrobial organism group analyses, there were three VMEs and two MEs. No organism-antibiotic pair analysis had more than a single VME or ME. Conclusions: P-AST, a component of the Guidance® UTI assay, demonstrates acceptable performance within the thresholds established by CLSI when compared against standard and reference methods for antibiotic susceptibility testing. Appropriate performance was established in both monomicrobial and polymicrobial specimens for five CLSI-defined groups of uropathogenic bacteria, against up to 20 antibiotics as clinically relevant to each organism group.