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

A duplex PCR to detect Bordetella pertussis and Bordetella parapertussis wa s 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-negatire resul ts 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 cult ure. Two patients Here positive for both B. pertussis and B. parapertussis. Patient records were reviewed to determine the validity of PCR-positive-cu lture-negative results. Forty-two of 39 patients who could be evaluated ful filled the criteria for a case definition of pertussis, with 32 patients be ing <1 year of age and having classical pertussis symptoms, The seven patie nts who did not fulfil the criteria were aged 7 to 55 Sears and had a persi stent cough for >2 weeks. The method Has also used to investigate a classro om outbreak in which B, pertussis culture was positive for 5 of 28 patients . II five culture-positive specimens were confirmed by PCR, and an addition al eight were positive by PCR. Of 25 patients from a suspected pertussis ou tbreak in a girls' dormitory, seven of seven specimens were negative for B. pertussis, although 13 of 25 patients were positive for B, pertussis immun oglobulin M (IgM) (2 of which produced equivocal IgA results, with 23 of 25 patients being negative). Five symptomatic patients were subsequently foun d to be positive (by IgM and particle agglutination assays) for Mycoplasma pneumoniae, demonstrating the value of PCR in rapidly excluding B. pertussi s 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 c ulture, indicating that a reassessment of the use of throat swabs should be considered, particularly for older patients, in contact tracing, and in si tuations in which specimen collection is difficult.