Nested duplex PCR to detect Bordetella pertussis and Bordetella parapertussis and its application in diagnosis of pertussis in nonmetropolitan Southeast Queensland, Australia

Abstract

A duplex PCR to detect Bordetella pertussis and Bordetella parapertussis was developed with the insertion sequences IS481 (B. pertussis) and IS1001 (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.

FIG. 1

Lanes (all values in parentheses are CFU per reaction mixture); a, 100-bp ladder; b, negative control; c to e, B. pertussis (1,000, 100, and 10, respectively); negative control; g to i, B. parapertussis (1,000, 100, and 10, respectively); j, negative control; k, B. pertussis (1,000) and B. parapertussis (1,000); l, B. pertussis (10) and B. parapertussis (1,000); m, B. pertussis (1,000) and B. parapertussis (10); n, B. pertussis (10) and B. parapertussis (10).