Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene

Douglas, Elaine; Coote, J. G.; Parton, R.; McPheat, William L.

doi:10.1099/00222615-38-2-140

Cited by 64 publications

(42 citation statements)

References 8 publications

(2 reference statements)

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“…In a comparison of different PCR assays used in pertussis vaccine studies, all assays provided an increased sensitivity for detection of pertussis cases by at least 70% in comparison to culture (23). Various PCR protocols have been developed that target different regions of the genome: insertion sequences IS481 and IS1001 (4,6,13,15,26,30), the pertussis toxin promoter region (6,15,20,26), the porin gene (5), and the adenylate cyclase gene (3). The achievements of the PCR assays targeting these different genes have not been widely compared.…”

mentioning

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

109

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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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mentioning

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

109

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“…Especially if negative B. pertussis-specific PCR results do not correspond to the clinical presentation, additional Information can be obtained by PCR of the adenylate cyclase gene, which is specific for all bordetellae species except B. avium (25).…”

Section: Discussionmentioning

confidence: 99%

Identification of Bordetella Pertussis in Nasopharyngeal Swabs Using the Polymerase Chain Reaction: Evaluation of Detection Methods

Lichtinghagen

Diedrich-Glaubitz²,

Horsten³

1994

CCLM

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Summary: A 183 base pairs or 153 base pairs DNA fragment from a repetitive region of the Bordetella pertussis genome was amplified in a polymerase chain reaction. The sensitivities of three different detection methods (Enzymun Test, silver stained polyacrylamide gel, ethidium bromide stained agarose gel) after amplification by polymerase chain reaction showed that both a one-time polymerase chain reaction (35 cycles) with Enzymun testing äs well äs a nested polymerase chain reaction with either of the electrophoresis methods have high levels of sensitivity for detection of the infectious organism in nasopharyngeal swabs. Smears from 53 children with whooping cough and from 50 children without infections were analysed, using these methods. 51 patients with whooping cough gave positive test results, while 2 of the sick patients and all the control children gave negative results.

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“…Serological tests to assist the diagnosis of pertussis are performed but, although more sensitive than culture, these usually only provide late, or retrospective diagnosis (Kerr & Matthews, 2000). To overcome these limitations, detection of B. pertussis DNA from nasopharyngeal aspirates (NPAs) and swabs (NPSs) has been described using PCR assays, including those targeting the promoter region of the gene encoding pertussis toxin S1 subunit (ptxA) (Houard et al, 1989;Mastrantonio et al, 1996), the insertion element IS481 (Glare et al, 1990), the adenylate cyclase gene (cyaA) (Douglas et al, 1993) and a region upstream of the outer-membrane porin gene (Li et al, 1994). However, few data are available to assess the relative utility of the various diagnostic approaches.…”

Section: Introductionmentioning

confidence: 99%

Laboratory diagnosis of pertussis infections: the role of PCR and serology

Fry¹,

Tzivra²,

Li³

et al. 2004

Journal of Medical Microbiology

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This study reports on practical laboratory aspects of pertussis diagnosis. PCR assays were applied to respiratory specimens obtained during a large study of infants (less than 5 months old) admitted to paediatric intensive care units (n ¼ 122), children (less than 15 years old) admitted to paediatric wards (n ¼ 16) and their household contacts (n ¼ 320). Estimation of antibodies to pertussis toxin and culture for Bordetella pertussis were attempted on specimens from the same patients, where available, and the overall utility of the diagnostic PCR assays was assessed by comparison to these results. A PCR assay for the human mitochondrial cytochrome oxidase (HMCO) gene was used for quality control of the extracted samples and an internal process control (IPC) was included in each sample to test for PCR inhibition. Four of 458 samples were considered unsuitable (three HMCO negative, one IPC negative) and excluded from further analyses. Positive PCR results were considered valid if they were either (i) positive for both of two B. pertussis gene targets (pertussis toxin S1 promoter and the insertion element IS481), i.e. consensus PCR positive, or (ii) repeatably positive in only one assay. Using these criteria, 52 of 454 (11 . 5 %) samples were considered as PCR positive for B. pertussis. Six of 356 samples were culture-positive for B. pertussis, 1/88 infants, 3/14 children and 2/254 contacts, giving an overall isolation rate of 1 . 7 %. Using these data, PCR gave an almost fivefold increase in diagnostic yield compared with culture (McNemar's test; P , 0 . 0001). Sera from 9/111 infants, 5/10 children and 14/210 contacts were positive. Serology and PCR results showed a high level of agreement (113/121) for infants and children. PCR demonstrated a significant improvement in diagnostic yield over culture. Serological testing also resulted in a significant increase in diagnostic yield compared to culture alone. PCR is a useful technique, but validity of results must be assured by careful control. Rapid diagnosis of B. pertussis infection particularly in infants by PCR, together with serological assays, can enhance surveillance systems for pertussis in all age groups.

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Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene

Cited by 64 publications

References 8 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

Identification of Bordetella Pertussis in Nasopharyngeal Swabs Using the Polymerase Chain Reaction: Evaluation of Detection Methods

Laboratory diagnosis of pertussis infections: the role of PCR and serology

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