Enrichment-Free Salmonella Quantification in Raw Poultry at the 1 and 10 CFU/g Decision Thresholds: A Multi-Laboratory Evaluation of the Pathotrak Concentrator with Real-Time PCR

Salmonella in raw poultry drives foodborne outbreaks and recalls, and process-control decisions increasingly turn on quantitative thresholds rather than presence/absence. Acting on a threshold in real time requires accurate quantification near the cutoff and a result within one work shift. Conventional enrichment-based enumeration meets neither: it adds hours of growth before detection, and the growth step biases the count toward faster-growing, unstressed cells. Near-threshold false positives also matter: products below the limit tests positive, triggering costly and avoidable holds and recalls. We evaluated a same-shift, enrichment-free workflow that couples the Pathotrak concentrator with real-time PCR (Bio-Rad iQ-Check Salmonella II) for quantifying Salmonella in raw poultry, with sample preparation in about 30 min instead of 6–24 h of enrichment. The evaluation spanned 313 independent inoculations across three laboratories, three calendar years (2023–2025), and multiple seasons, technicians, instruments, and chicken sources. A latent-truth framework propagated inoculation uncertainty into sensitivity (Se) and specificity (Sp) estimates and made the half-log gray zone around each cutoff tractable. At 10 CFU/g, the workflow achieved Se 100% (20/20; 95% CI 83.2–100.0%) for samples at least 0.5 log10 above the threshold and Sp 94.4% (185/196; 95% CI 90.2–97.2%) below it; within the half-log gray zone, classification was Se 88.6% (95% CI 68.8–99.1%)/Sp 76.5% (95% CI 62.1–88.1%) (n = 77). In a cross-study comparison, gray-zone performance at 10 CFU/g fell within the range reported for MPN, with inoculation strain, chicken source, and plate-count method differing by site. At 1 CFU/g, the workflow returned a calibrated quantitative result within a single shift, which, to our knowledge, no enrichment-free real-time-PCR workflow has previously achieved without an incubation step. Samples more than 0.5 log10 CFU/g above or below a threshold were classified correctly in 205/216 (94.9%) cases at 10 CFU/g and 109/117 (93.2%; 95% CI 87.0–97.0%) at 1 CFU/g, with classification uncertainty confined to the gray zone (Se 63.2% (95% CI 49.9–76.0%)/Sp 66.5% (95% CI 54.2–77.9%), n = 166) as expected at low copy number (Salmonella genome copies estimated by qPCR).