Dataset Information

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-pertussis Bordetella species in our surveilled area.

SUBMITTER: Valero-Rello A

PROVIDER: S-EPMC6322476 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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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.

Valero-Rello Ana A Henares Desiree D Acosta Lesly L Jane Mireia M Jordan Iolanda I Godoy Pere P Muñoz-Almagro Carmen C

Journal of clinical microbiology 20190102 1

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 stra ...[more]

PMID: 30404946

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Project description:A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.

Dataset Information

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.

Publications

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.

Similar Datasets

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