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c While PCR is the most common method used for detecting Bordetella pertussis in the United States, most laboratories use insertion sequence 481 (IS481), which is not specific for B. pertussis; therefore, the relative contribution of other Bordetella species is not understood. The objectives of this study were to evaluate the proportion of other Bordetella species misidentified as B. pertussis during a period of increased pertussis incidence, determine the level of agreement in Bordetella species detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDC's PCR assay when using a different PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories for B. pertussis and B. parapertussis detection. Every fifth specimen positive for IS481 and/or IS1001 with cycle threshold (C T ) values of <35 was sent to CDC for PCR testing that identifies Bordetella species. Specimens with indeterminate or negative results in the CDC PCR were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (n ‫؍‬ 630). Of the specimens with different identifications (n ‫؍‬ 125), 79.2% (n ‫؍‬ 99) were identified as indeterminate B. pertussis at CDC. Overall, 0.66% (n ‫؍‬ 5) of the specimens were identified as B. holmesii or B. bronchiseptica at CDC. Of 115 specimens with indeterminate or negative results, 46.1% (n ‫؍‬ 53) were B. pertussis positive when tested by an alternate master mix, suggesting a possible increase in assay sensitivity. This study demonstrates good agreement between the two U.S. commercial laboratories and CDC and little misidentification of Bordetella species during the 2012 U.S. epidemic. P ertussis, commonly known as whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States. The number of reported cases has increased in the last 10 years, with more than 48,000 cases reported in 2012 (1, 2). Factors that may contribute to the resurgence of pertussis include waning immunity following vaccination and bacterial adaptation. Improved detection and diagnosis may also have increased due to greater public health awareness and the use of high-sensitivity laboratory diagnostic techniques such as PCR. PCR assays targeting the insertion sequence 481 (IS481), which is present in multiple copies (about 200 to 250 copies) in the Bordetella pertussis genome (3-5), are the most commonly used pertussis diagnostics in the United States. However, the specificity of these assays is compromised because the same target sequence is also present in other Bordetella species such as B. holmesii, which causes pertussis-like illness, and B. bronchiseptica, which occasionally infects humans (4, 6). Consequently, although high IS481 cycle threshold (C T ) values may be indicative of low levels of DNA from a patient with pertussis, they may also indicate a misidentified specimen from a patient infected with another Bordetella species or f...