We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS481 for the detection of Bordetella pertussis in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true B. pertussis infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of B. pertussis.

After our manuscript comparing the performance of PCR, culture, and direct fluorescent-antibody testing for Bordetella pertussis (3) went to press, we obtained six isolates of Bordetella holmesii for the purpose of testing with our B. pertussis IS481 PCR assay. This request for isolates was prompted by the recent report by Yih et al. ( 6) on the isolation of B. holmesii from nasopharyngeal (NP) specimens from patients with pertussis-like cough. Previously, B. holmesii has been associated with septicemia in immunocompromised patients (5) and was isolated from the sputum of patients with respiratory failure (4). Yih et al. isolated B. holmesii infrequently from NP specimens during their evaluation (0.26% positivity rate, compared to 6.6% positivity rate for B. pertussis) (6).B. holmesii isolates (kindly provided by H. George, Massachusetts Department of Public Health) were grown on Trypticase soy agar plates containing 5% sheep blood for 48 h at 35°C. Colony picks were suspended in sterile water and were tested with the B. pertussis PCR assay as previously described (3). Initially, the quantity of organisms added to PCRs was not determined, but it was estimated to be at least 100 to 1,000 CFU per reaction. All six B. holmesii isolates generated strong positive results with the B. pertussis IS481 PCR assay. To evaluate the limit of detection, suspensions of two B. holmesii isolates were prepared, and cell concentrations were estimated based on McFarland turbidimetric standards. Aliquots of serial dilutions were tested with the B. pertussis PCR assay. The analytical sensitivity ranged from 0.06 to 0.3 CFU per PCR, which is similar to the sensitivity we previously reported for B. pertussis (3).B. pertussis PCR assays that target the IS481 repetitive element may cross-react with B. holmesii. Our PCR assay utilizes previously described primers and probe. The sequences of the upstream and downstream primers were previously described by Glare et al. (1) and He et al. (2), respectively. The sequence of our capture probe is identical to that of the downstream primer used by Glare et al. (1). We have not determined whether actual NP specimens from patients infected or colonized with B. holmesii would generate a positive result with our B. pertussis PCR test. We have not isolated B. holmesii from NP swab specimens in our laboratory. However, our Regan-Lowe transport medium contains 40 g of cephalexin per ml, to which B. holmesii is reportedly susceptible (E. Mazengia, H.