Multiplex Polymerase Chain Reaction-Based Assay for the Detection Ofbordetella Pertussis in Nasopharyngeal Swab Specimens.† 1108

Wadowsky, Robert M.; Libert, Therese; Ehrlich, Garth D.; Kingsley, L.; Michaels, Richard H.

doi:10.1203/00006450-199604001-01130

Cited by 11 publications

(14 citation statements)

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“…B. pertussis is a particularly labile organism, and most diagnostic laboratories are unable to culture the patient's material directly from the patient. In this study culture sensitivity was 38%, but others have reported rates of 15.2% (13) and 73.4% (33). The difference in sensitivity rates can be explained by differences in transportation to the laboratory, differences in when the sample is inoculated onto agar, and also the age of the patients.…”

Section: Discussionmentioning

confidence: 56%

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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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Section: Discussionmentioning

confidence: 56%

“…PCR has repeatedly been shown to be more sensitive than culture and direct fluorescent-antibody assay for detection of B. pertussis (7,13,23,29,30,33). However, analysis of discrepant results and assessment of clinical performance of PCR have been carried out less frequently (13,33).…”

Section: Discussionmentioning

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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“…As is the case for other fastidious organisms, PCR offers an attractive alternative for detecting B. pertussis and B. parapertussis in clinical specimens (1,4,5,7,15,17,21,26). Previously evaluated B. pertussis target regions include insertion sequences (IS), repeat elements, the pertussis toxin promoter region, the adenylate cyclase gene, and the porin gene.…”

Section: Detection Of Bordetella Holmesii By a Real-time Pcr Assay Tamentioning

confidence: 99%

Real-Time PCR Assay Targeting IS 481 of Bordetella pertussis and Molecular Basis for Detecting Bordetella holmesii

et al. 2001

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Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported.Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.Bordetella pertussis is the causative agent of whooping cough, an infectious disease that occurs worldwide with a high prevalence among young, unvaccinated infants (2,3,6,19) and is recently resurgent in highly vaccinated populations. Related species, including B. parapertussis, B. bronchiseptica, and the more recently described B. holmesii (27), may also cause a pertussis-like syndrome in humans. Laboratory diagnosis of pertussis is traditionally based primarily on culture, which is highly specific but is maximally sensitive only in the initial phases of disease (10,14). Furthermore, culturing depends on specimen quality and laboratory expertise and requires special media, extended incubation periods of 7 days or more, and confirmation by biochemical or antibody reagent tests. Diagnostic serology can be highly sensitive and more rapid than culture (14), but no serologic assay has been approved for diagnostic use in the United States because no diagnostic criterion has been widely accepted and no method has been validated between laboratories.Thus, there is a need for more rapid and sensitive diagnostic methods that have high positive predictive value, especially in the early stages of disease. As is the case for other fastidious organisms, PCR offers an attractive alternative for detecting B. pertussis and B. parapertussis in clinical specimens (1,4,5,7,15,17,21,26). Previously evaluated B. pertussis target regions include insertion sequences (IS), repeat elements, the pertussis toxin promoter region, the adenylate cyclase gene, and the porin gene. IS481 is present in the B. pertussis genome at 80 (22) to 100 (5, 18) copies, and PCR assays targeting IS481 have been evaluated for sensitivity and specificity in several laboratories over the last few years. Therefore, an internal region of IS481 was selected as the B. pertussis target (18) while a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) duplex PCR for B. pertussis and B. parapertussis was being developed. The previously described (5) forward primer BP-1 (5Ј GAT TCA ATA GGT TGT ATG CAT GGT T 3Ј and the slightly modified reverse primer BP-2 (5Ј TTC AGG CAC ACA AAC TTG ATG GGC G 3Ј), corresponding to bp 12 to 36 and 192 to 167 (GenBank M22031) of IS481, respectively, were used for amplification. A pair of fluorescence-labeled hybridization probes, BP-HP-3 (5Ј TCG CCA ACC CCC CAG TTC ACT CA-FAM 3Ј) and BP-HP-4 (5Ј LC red 640-AGC CCG GCC GGA TGA ACA CCC-3Ј-phosphate), corresponding to bp 66 to 88 and 92 to 112 (GenBank M22031) of IS481, respecti...

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“…Sensitive and specific PCR assays have been developed by several investigators to amplify and detect B. pertussis DNA (10,29,35,36). A primary advantage of this diagnostic method is the rapid turnaround time, which typically amounts to a few hours (2).…”

mentioning

confidence: 99%

Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis

et al. 2002

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Multiplex Polymerase Chain Reaction-Based Assay for the Detection Ofbordetella Pertussis in Nasopharyngeal Swab Specimens.† 1108

Cited by 11 publications

References 0 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

Real-Time PCR Assay Targeting IS 481 of Bordetella pertussis and Molecular Basis for Detecting Bordetella holmesii

Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis

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