Abstract
Objective: We evaluated the diagnostic accuracy of a novel CRISPR‑Cas12a guided rapid test for detecting Salmonella enterica serovar Typhi, including extensively drug‑resistant (XDR) strains, in comparison with gold‑standard polymerase chain reaction (PCR). We additionally assessed usability in low‑resource laboratories and the impact on clinical decision‑making and antibiotic stewardship in a tertiary hospital setting in Punjab, Pakistan.
Methods: In this multicenter diagnostic clinical trial, we enrolled febrile patients with suspected enteric fever across three clinical centers. Blood samples were processed concurrently using (1) blood culture with PCR confirmation targeting S. Typhi–specific genes; and (2) the CRISPR‑Cas12a rapid assay integrating isothermal amplification with Cas12a detection. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated against PCR as reference. Laboratory technicians in low‑resource settings scored usability metrics via standardized questionnaires.
Results: Out of N=1,200 evaluable samples, the CRISPR‑Cas12a test demonstrated a sensitivity of 93.5% (95% CI 90.8–95.8) and specificity of 95.2% (92.7–97.0) compared with PCR. Diagnostic performance was maintained in isolates characterized as XDR (n=312). Usability assessments indicated high operator agreement on ease of use (mean score 4.5/5) and minimal equipment requirements. Implementation of the rapid test reduced time to targeted antibiotic therapy by a median of 24 h and was associated with improved antibiotic stewardship indicators.
Conclusion: The CRISPR‑Cas12a rapid assay exhibited high diagnostic accuracy for both typical and XDR S. Typhi infections and proved feasible in low‑resource laboratories. Adoption of this assay could enhance early diagnosis, rational antibiotic use, and patient outcomes in high‑burden settings.