This study aimed to validate a comprehensive diagnostic protocolbased on real-time PCR for the rapid detection and identification ofBordetella per-tussis,Bordetella parapertussis, andBordetella holmesii, as well as its implementationin the diagnostic routine of a reference children’s hospital. The new algorithm in-cluded a triplex quantitative PCR (qPCR) targeting IS481gene (inB. pertussis,B. hol-mesii, and someBordetella bronchisepticastrains), pIS1001(B. parapertussis-specific)andrnaseP as the human internal control. Two confirmatory singleplex tests forB.pertussis(ptxA-Pr) andB. holmesii(hIS1001) were performed if IS481was positive. An-alytical validation included determination of linear range, linearity, efficiency, preci-sion, sensitivity, and a reference panel with clinical samples. Once validated, the newalgorithm was prospectively implemented in children with clinical suspicion ofwhooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomicequivalents/ml of sample for IS481(onB. pertussis), pIS1001and hIS1001, and 777.9forptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay vari-abilities were 3% and 5%, respectively. Among 566 samples analyzed,B. pertus-sis,B. holmesii, andB. parapertussiswere detected in 11.1%, 0.9% (only in females 4 years old), and 0.2% of samples, respectively. The new algorithm proved to be auseful microbiological diagnostic tool for whooping cough, demonstrating a low rateof other non-pertussis Bordetellaspecies in our surveilled area

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