Bordetella pertussis and Bordetella parapertussis, Molecular Detection, PCR
Preferred diagnostic test for the detection of Bordetella pertussis and/or Bordetella parapertussis
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Pertussis is an infectious respiratory disease caused by the Bordetella pertussis bacterium. Pertussis afflicts unvaccinated individuals, and those in whom immunity has waned. Infants are at particular risk for severe disease and complications. Adults present with a prolonged chronic cough illness. Bordetella parapertussis may cause a similar illness, especially in children; however, the symptoms are less severe and of shorter duration than those associated with Bordetella pertussis.
The wide prevalence of pertussis and its changing epidemiology highlight the need for sensitive and rapid methods for diagnostic testing. Clinical diagnosis of pertussis is complicated because the characteristic cough (whoop) is rarely seen in infant and adult patients.
Several diagnostic methods are available, but many lack sensitivity and/or require repeat testing or extended incubation times for test results. The reference method has traditionally been direct culture of the organism from nasopharyngeal secretions. However, due to the fastidious nature of the organism, recovery by culture is difficult, as the organism is susceptible to environmental exposure (change in temperature and drying) and has specific growth requirements. Direct fluorescent antibody testing of material collected by a nasopharyngeal swab lacks sensitivity.(1) PCR is the Mayo-recommended test for detection of Bordetella pertussis and parapertussis in patients suspected of having active, untreated pertussis. PCR is preferred over culture because it is faster and demonstrates improved sensitivity
Additional information regarding pertussis PCR testing has been published by the CDC and can be found at URL: http://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
A positive result indicates the presence of DNA from Bordetella pertussis or Bordetella parapertussis. In some cases, a patient may test positive for both Bordetella pertussis and Bordetella parapertussis. Cross-reactivity with Bordetella holmesii and Bordetella bronchiseptica may occur with the Bordetella pertussis assay (see Cautions).
A negative result indicates the absence of detectable Bordetella pertussis or Bordetella parapertussis DNA in the specimen but does not negate the presence of organism or active or recent disease (known inhibition rate of <1%) and may occur due to inhibition of PCR, sequence variability underlying primers and/or probes, or the presence of Bordetella pertussis or Bordetella parapertussis in quantities less than the limit of detection of the assay.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Cross-reactivity with Bordetella holmesii may occur with the Bordetella pertussis PCR assay. The prevalence of Bordetella holmesii is relatively low, with positivity in <1% of nasopharyngeal swabs.(2) Bordetella holmesii has, however, been associated with pertussis-like symptoms.(2)
Cross-reactivity of the Bordetella pertussis assay has been demonstrated with a limited number of Bordetella bronchiseptica isolates. The prevalence of the insertion sequence target, IS481, has been reported to be between 1% and 5% in Bordetella bronchiseptica isolates.
This assay is not recommended for screening asymptomatic individuals who may carry Bordetella pertussis or parapertussis
This assay is not recommended for follow up of patients previously diagnosed with pertussis (ie, as a test of cure).
Due to recently mandated FDA regulations concerning the sale of analyte specific reagents (ASR), Roche no longer produces Bordetella reagents that were used in our previous assay.(3) In response to this, we developed a rapid real-time multiplex reaction LightCycler PCR assay to detect and differentiate Bordetella pertussis and Bordetella parapertussis (LightCycler BORD-T) in nasopharyngeal swabs/washings. We compared its performance to the previous LightCycler ASR PCR system (LightCycler BORD ASR), which used the Roche ASR. Similar to the LightCycler BORD ASR, the LightCycler BORD-T assay targets the multicopy insertion gene sequences IS481 and IS1001 of Bordetella pertussis and parapertussis, respectively. Results of the LightCycler BORD-T assay were compared to results of the LightCycler BORD ASR assay on 374 nasopharyngeal swabs and washings submitted for Bordetella testing. Fifty-four specimens were positive (48 Bordetella pertussis and 6 Bordetella parapertussis) and 314 specimens were negative by both PCR assays. Five nasopharyngeal specimens were positive for Bordetella pertussis or Bordetella parapertussis by the LightCycler BORD-T and negative by the LightCycler BORD ASR. One nasopharyngeal specimen was positive for Bordetella pertussis by the LightCycler BORD ASR but negative by the LightCycler BORD-T assay. The LightCycler BORD-T assay demonstrated 98% (368/374) agreement with the BORD ASR. Thirty respiratory microorganisms (pathogenic and non-pathogenic), including 4 different Bordetella species, were tested with the LightCycler BORD-T. Bordetella pertussis and Bordetella parapertussis were positive as expected and Bordetella holmesii was detected at the same melt temperature as the Bordetella pertussis IS481 gene target. Bordetella holmesii cannot be distinguished from Bordetella pertussis by the LightCycler BORD-T assay. All other results were negative. The analytical sensitivity of the LightCycler BORD-T was 1 target/mcL for nasopharyngeal swabs and 10 targets/mcL for nasopharyngeal wash/aspirates.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. She RC, Billetdeaux E, Phansalkar AR, et al: Limited applicability of direct fluorescent-antibody testing for Bordetella sp. and Legionella sp. specimens for the clinical microbiology laboratory. J Clin Microbiol 2007;45:2212-2214
2. Guthrie JL, Robertson AV, Tang P, et al: Novel duplex real-time PCR assay detects Bordetella holmesii in specimens from patients with pertussis-like symptoms in Ontario, Canada. J Clin Microbiol 2010;48:1435-1437
3. Sloan LM, Hopkins MK, Mitchell PS, et al: Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens. J Clin Microbiol 2002;40:96-100
4. Dragsted DM, Dohn B, Madsen J, et al: Comparison of culture and PCR for detection of Bordetella pertussis and Bordetella parapertussis under routine laboratory conditions. J Med Microbiol 2004;53:749-754
5. Antila M, He Q, de Jong C, et al: Bordetella holmesii DNA is not detected in nasopharyngeal swabs from Finnish and Dutch patients with suspected pertussis. J Med Microbiol 2006;55:1043-1051