Bordetella pertussis PCR: simultaneous targeting of signature sequences

Qin, Xuan; Turgeon, David; Ingersoll, Brian P.; Monsaas, Peter W.; Lemoine, Christina J.; Tsosie, Treva; Stapp, Lynn O; Abe, Patrick M.

doi:10.1016/s0732-8893(02)00405-4

Cited by 18 publications

(23 citation statements)

References 20 publications

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“…The achievements of the PCR assays targeting these different genes have not been widely compared. The insertion sequences have been found to be more sensitive than the pertussis toxin promoter (26), but other workers have shown comparable sensitivities for the insertion sequences and the porin gene (5) and the insertion sequences and the pertussis toxin promoter (25). The copy number of the insertion sequence IS481 in B. pertussis is approximately 100, the copy number of the insertion sequence IS1001 in B. parapertussis is 20 to 21 (32), and for B. holmesii the copy number is unknown.…”

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confidence: 99%

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Templeton¹,

Scheltinga²,

Zee³

et al. 2003

J Clin Microbiol

112

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PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

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confidence: 99%

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Templeton¹,

Scheltinga²,

Zee³

et al. 2003

J Clin Microbiol

112

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“…The use of multiple target sequences serves as an important analytical control for the PCRs. While it is possible to control for reagent and instrument function by setting up standard positive and negative controls, the possibility of technical problems or operator error cannot be readily assessed unless multiple reactions are used (14).…”

Section: Discussionmentioning

confidence: 99%

Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

Qin¹,

et al. 2003

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Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.Pseudomonas aeruginosa is the most significant pathogen affecting patients with cystic fibrosis (CF). It is often acquired in the first few years of life and causes chronic airway infections, which ultimately result in death. While P. aeruginosa is not thought of as an organism that is difficult to identify in the microbiology laboratory, CF isolates often have unique phenotypes, including the loss of pigment production, synthesis of rough lipopolysaccharide devoid of O side chains, and development of mucoidy (18). While the presence of mucoidy may be helpful in identifying CF isolates of P. aeruginosa, some of the other characteristics may make these isolates more difficult to identify. Both commercial methods of identification and standard biochemical testing may be lengthy and inaccurate (10, 11). In addition, CF patients often have other nonfermenting gram-negative bacilli isolated from their respiratory secretions, making rapid isolation and identification of P. aeruginosa even more difficult (1, 7). While sequencing 16S rRNA genes in CF isolates of P. aeruginosa and other gram-negative bacilli has utility (6), it may be expensive and time-consuming.The Cystic Fibrosis Foundation-funded Therapeutic Development Network (...

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“…49 Sensitivity and specificity of PCR depend on the primers used, and various combined techniques have been reported to enhance the performance of this method. 50,51 However, the sensitivity of PCR decreases with the duration of symptoms, since the method is based on the detection of the microorganism.…”

Section: Diagnosismentioning

confidence: 99%

Diagnosis and management of pertussis

Tozzi¹

2005

Canadian Medical Association Journal

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PERTUSSIS IS INCREASING IN FREQUENCY among children too young to be vaccinated and among adolescents and adults. This increase is due mainly to waning immunity among vaccinated individuals, who become susceptible during adolescence and adulthood and maintain the circulation of Bordetella pertussis. Infants are at highest risk of severe illness requiring hospital admission, complications and death. The clinical presentation in adolescents, adults and vaccinated individuals may be atypical, with paroxysmal cough of short duration or simply a persistent cough. Culture and polymerase chain reaction may be used to identify B. pertussis infection, but their sensitivity is high only in the early phase of the disease. Serologic tests are not standardized for the diagnosis of pertussis, and their clinical application is limited. Erythromycin is still considered in some countries to be the “gold standard” for therapy and prophylaxis; however, azithromycin and clarithromycin seem equally efficacious and are associated with fewer side effects

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Bordetella pertussis PCR: simultaneous targeting of signature sequences

Cited by 18 publications

References 20 publications

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

Diagnosis and management of pertussis

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