Nested Duplex PCR To Detect Bordetella pertussis and Bordetella parapertussis and Its Application in Diagnosis of Pertussis in Nonmetropolitan Southeast Queensland, Australia

Abstract

ABSTRACT A duplex PCR to detect Bordetella pertussis and Bordetella parapertussis was developed with the insertion sequences IS 481 ( B. pertussis ) and IS 1001 ( B. parapertussis ) and evaluated with specimens from 520 consecutive patients presenting with possible pertussis. No culture-positive–PCR-negative results occurred, giving the method a sensitivity of 100%. For B. pertussis , 58 of 520 patients (11.2%) were positive by PCR compared to 17 of 520 patients positive (3.3%) by culture. For B. parapertussis , 7 of 520 patients (1.3%) were positive by PCR compared to 2 of 520 patients positive (0.4%) by culture. Two patients were positive for both B. pertussis and B. parapertussis . Patient records were reviewed to determine the validity of PCR-positive–culture-negative results. Forty-two of 49 patients who could be evaluated fulfilled the criteria for a case definition of pertussis, with 32 patients being <1 year of age and having classical pertussis symptoms. The seven patients who did not fulfil the criteria were aged 7 to 55 years and had a persistent cough for >2 weeks. The method was also used to investigate a classroom outbreak in which B. pertussis culture was positive for 5 of 28 patients. All five culture-positive specimens were confirmed by PCR, and an additional eight were positive by PCR. Of 25 patients from a suspected pertussis outbreak in a girls’ dormitory, seven of seven specimens were negative for B. pertussis , although 13 of 25 patients were positive for B. pertussis immunoglobulin M (IgM) (2 of which produced equivocal IgA results, with 23 of 25 patients being negative). Five symptomatic patients were subsequently found to be positive (by IgM and particle agglutination assays) for Mycoplasma pneumoniae , demonstrating the value of PCR in rapidly excluding B. pertussis infection in an outbreak situation. Twenty-two of 71 (30.1%) throat swabs were positive by PCR compared to 2 of 71 (2.8%) throat swabs positive by culture, indicating that a reassessment of the use of throat swabs should be considered, particularly for older patients, in contact tracing, and in situations in which specimen collection is difficult.