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.
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.
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DOI：
10.1128/jcm.01231-18
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发表时间：
2019-01
影响因子：
9.4
通讯作者：
Muñoz-Almagro C
中科院分区：
文献类型：
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作者：
Valero-Rello A;Henares D;Acosta L;Jane M;Jordan I;Godoy P;Muñoz-Almagro C
本研究旨在验证基于实时PCR的快速检测和鉴定百日咳博德特氏菌、副百日咳博德特氏菌和霍氏博德特氏菌的综合诊断方案，以及其在参考儿童医院诊断常规中的实施。新算法包括靶向IS 481基因的三重定量PCR（qPCR）（在B.百日咳，B. B. holmesii和一些支气管败血波氏杆菌菌株）、pIS 1001（B.副百日咳特异性）和RNA酶P作为人内部对照。本研究旨在验证基于实时PCR的快速检测和鉴定百日咳博德特氏菌、副百日咳博德特氏菌和霍氏博德特氏菌的综合诊断方案，以及其在参考儿童医院诊断常规中的实施。新算法包括靶向IS 481基因的三重定量PCR（qPCR）（在B.百日咳，B. B. holmesii和一些支气管败血波氏杆菌菌株）、pIS 1001（B.副百日咳特异性）和RNA酶P作为人内部对照。B的两项确证性单重检测。百日咳（ptxA-Pr）和B.如果IS 481为阳性，则进行Holmesii（hIS 1001）。分析验证包括测定线性范围、线性、效率、精密度、灵敏度和临床样本的参考样本组。一旦得到验证，新算法将在12个月内在Sant Joan de Deu医院（西班牙巴塞罗那）临床疑似百日咳的儿童中前瞻性实施。对于IS 481（在B上），获得的检测下限分别为4.4、13.9和27.3基因组当量/ml样品。pIS 1001和hIS 1001的qPCR效率为86.0%至96.9%。批内和批间变异性分别<3%和<5%。在分析的566个样品中，B.百日咳，B. B. holmesii和B.副百日咳检出率分别为11.1%、0.9%（仅>4岁的女性）和0.2%。新算法被证明是百日咳的一种有用的微生物诊断工具，表明在我们的监测区域中其他非百日咳博德特氏菌属的比例很低。
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. 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.