Validation and Implementation of a Diagnostic Algorithm for DNA Detection of Bordetella pertussis, B. parapertussis, and B. holmesii in a Pediatric Referral Hospital in Barcelona, Spain.

This study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification of , , and , as well as its implementation in the diagnostic routine of a reference children's hospital. The new algorithm included a triplex quantitative PCR (qPCR) targeting IS gene (in , , and some strains), pIS (-specific) and P as the human internal control. Two confirmatory singleplex tests for (-Pr) and (hIS) were performed if IS was positive. Analytical validation included determination of linear range, linearity, efficiency, precision, sensitivity, and a reference panel with clinical samples. Once validated, the new algorithm was prospectively implemented in children with clinical suspicion of whooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over 12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomic equivalents/ml of sample for IS (on ), pIS and hIS, and 777.9 for -Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed, , , and were detected in 11.1%, 0.9% (only in females >4 years old), and 0.2% of samples, respectively. The new algorithm proved to be a useful microbiological diagnostic tool for whooping cough, demonstrating a low rate of other non- species in our surveilled area.