Bordetella pertussis and Bordetella parapertussis, Molecular Detection, PCR
Preferred diagnostic test for the detection of Bordetella pertussis or Bordetella parapertussis
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Bordetella pertussis is the highly contagious etiological agent of pertussis or whooping cough. Bordetella parapertussis causes a similar, but generally less severe illness. Despite vaccination efforts, Bordetella pertussis remains common in the United States, underscoring the need for effective diagnostic tests. In the United States, pertussis is most common in the late summer months. Pertussis vaccination does not prevent Bordetella parapertussis infection, which generally occurs in a younger age group than disease caused by Bordetella pertussis. Diagnosis of pertussis is based on having a high clinical index of suspicion for the infection, along with confirmation by laboratory testing. Laboratory testing methods include nucleic acid amplification tests (eg, PCR), serology, culture and direct fluorescent antibody testing. Culture and direct fluorescent antibody testing are limited by low sensitivity, rendering nucleic acid amplification tests and serology the tests of choice.
The Centers for Disease Control and Prevention recommends PCR testing for patients suspected of having acute pertussis. Bordetella pertussis PCR detects roughly twice as many cases as culture. Bordetella pertussis DNA can be detected up to 4 weeks, or longer (up to 8 weeks in our experience),(1) after symptom onset. However, over time, the amount of Bordetella pertussis and Bordetella parapertussis DNA will diminish, rendering the assay less sensitive. A serologic response to Bordetella pertussis is typically mounted by 2 weeks following infection and, therefore, detection of IgG-class antibodies to pertussis toxin (PT), which is only produced by Bordetella pertussis, can be a useful adjunct for diagnosis at later stages of illness at a time when the amount of Bordetella pertussis may be below the limit of detection of the PCR assay.
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 and 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. Additionally, patients presenting late after symptom onset may test negative; in such cases, testing for Bordetella pertussis antibody, IgG, in serum may be considered.
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) Please note that Bordetella holmesii has 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).
Some Bordetella pertussis acellular vaccines (ie, Pentacel, Daptacel, Adacel) contain PCR detectable DNA. Contamination of specimens with vaccine can cause false-positive Bordetella pertussis PCR results. Specimens should not be collected or processed in areas that are exposed to Bordetella pertussis vaccine material.
The assay targets the multicopy insertion gene sequences, IS481 and IS1001, of Bordetella pertussis and Bordetella parapertussis, respectively. This assay was previously performed using analyte specific reagents from Roche Diagnostics(3); these reagents are no longer available. The assay was revalidated using probes and primers with the same sequence, but provided by an alternate vendor. Performance of the new assay was then compared to the previous assay, which used the Roche analyte specific reagents, using 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 assays. Five nasopharyngeal specimens were positive for Bordetella pertussis or Bordetella parapertussis by the new assay and negative by the old assay. One nasopharyngeal specimen was positive for Bordetella pertussis by the old assay but negative by the new assay. Overall, there was 98% (368/374) agreement between the 2 assays. Bordetella holmesii cannot be distinguished from Bordetella pertussis by the assay. The analytical sensitivity of the assay is 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. Theofiles AG, Cunningham SA, Chia N, et al: Pertussis outbreak, southeastern Minnesota, 2012. Mayo Clin Proc 2014 Oct;89(10):1378-1388
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