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

Abstract

This study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii, 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 IS481 gene (in B. pertussis, B. holmesii, and some Bordetella bronchiseptica strains), pIS1001 (B. parapertussis-specific) and rnase P as the human internal control. Two confirmatory singleplex tests for B. pertussis (ptxA-Pr) and B. holmesii (hIS1001) were performed if IS481 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 IS481 (on B. pertussis), pIS1001 and hIS1001, and 777.9 for ptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed, B. pertussis, B. holmesii, and B. parapertussis 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-pertussisBordetella species in our surveilled area.

Keywords: B. holmesii; B. parapertussis; Bordetella pertussis; real-time PCR; whooping cough.

FIG 1

Seasonal distribution of microbiologically confirmed whooping cough cases by etiological agent (n = 566), includes one coinfection of B. holmesii and B. pertussis (*).

FIG 2

Age distribution of microbiologically confirmed cases and proportion of Bordetella species (n = 566). Includes one coinfection of B. holmesii and B. pertussis (*).