Evaluation of Level of Agreement in Bordetella Species Identification in Three U.S. Laboratories during a Period of Increased Pertussis

Abstract

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 (CT) 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.

FIG 1

Scatterplots of the commercial laboratory cycle threshold (C_T) values versus CDC IS481 C_T values. Specimens with undetected IS481 were assigned a C_T value of 46. The dashed line is the line of identity, and the solid black line is the Deming regression line. (A) Commercial laboratories A plus B C_T values versus CDC IS481 C_T values (n = 702). The Deming regression line was y = 1.29x − 6.26. (B) Laboratory A C_T values versus CDC IS481 C_T values (n = 414). The Deming regression line was y = 1.25x − 4.19. (C) Laboratory B C_T values versus CDC IS481 C_T values (n = 288). The Deming regression line was y = 1.32x − 8.86.