Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost

Tilley, Peter; Kanchana, M. V.; Knight, I. G.; Blondeau, Joseph M; Antonishyn, Nick A.; Deneer, Harry

doi:10.1016/s0732-8893(00)00117-6

Cited by 38 publications

(27 citation statements)

References 27 publications

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“…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%

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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

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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Section: Discussionmentioning

confidence: 99%

“…Laboratory diagnosis is traditionally based on culture, which is regarded as the "gold standard" and is highly specific but whose sensitivity in comparison to PCR or serology varies between 7 and 36% (13,29,31). Furthermore, culturing depends on specimen quality, rapid transportation, and laboratory expertise.…”

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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“…Laboratory confirmation by culture and isolation of the causative organism is generally considered to be the gold standard for the diagnosis of the majority of bacterial infections, because its specificity is effectively 100 %. B. pertussis is a fastidious and fragile organism and it is now widely recognized that reliance on culture alone is likely to lead to significant under-reporting of cases of pertussis infection (Müller et al, 1997;SchmidtSchläpfer et al, 1997;Tilley et al, 2000).…”

Section: Resultsmentioning

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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“…5). Direct examination of smears by the monoclonal fluorescent-antibody technique (14) is successful for the detection of B. pertussis, although rates of false-negative results as high as 50% have been reported (5,20). Also, the use of UV microscopy is labor-intensive, and accuracy typically falters in times of large outbreaks, when it is needed the most (4).…”

Section: Discussionmentioning

confidence: 99%

“…Like other endotoxins, B. pertussis LPS is a durable molecule that can easily withstand the conditions encountered during handling and transport. The detection of antibody to LPS is a good strategy for the diagnosis of B. pertussis infection, but the method could be improved to make it easier to use and a more sensitive reagent is needed (20).…”

mentioning

confidence: 99%

Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

Tang

Peppler

Irvin

et al. 2004

Clin Vaccine Immunol

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A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solutionphase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.Whooping cough (pertussis) is a vaccine-preventable disease caused by the gram-negative bacterium Bordetella pertussis. Despite required vaccination in most countries, the incidence of whooping cough is still unacceptably high and has recently emerged in the adolescent age group (2, 10). Accurate diagnosis of B. pertussis is essential to proper surveillance of whooping cough, and improved diagnostic methods are desirable.One of the present methods for the diagnosis of B. pertussis infection is a direct fluorescent-antibody assay that uses a fluorescence-labeled monoclonal antibody (MAb) directed against the lipopolysaccharide (LPS) in the outer membrane of the bacterium (14). Unlike many other bacteria, B. pertussis produces one predominant antigenic type of LPS molecule (17), making it a good specific target for immunodiagnosis. Like other endotoxins, B. pertussis LPS is a durable molecule that can easily withstand the conditions encountered during handling and transport. The detection of antibody to LPS is a good strategy for the diagnosis of B. pertussis infection, but the method could be improved to make it easier to use and a more sensitive reagent is needed (20).Here we report on the development and characterization of a bispecific MAb (bsMAb) against horseradish peroxidase (HRPO) and B. pertussis LPS that could be used for enzymebased detection of soluble LPS antigen and whole B. pertussis bacteria in clinical samples, immunochemical structural studies, and serological characterization of B. pertussis LPS, with some potential advantage over present MAb-or polyclonal antibody-labeled immunoassays. In particular, ...

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Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost

Cited by 38 publications

References 27 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

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

Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

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