Harmonization of Bordetella pertussis real-time PCR diagnostics in the US, 2012.

Williams MM1, Taylor TH Jr2, Warshauer DM3, Martin MD2, Valley AM3, Tondella ML2.

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Abstract

Real-time (rt-) PCR is an important diagnostic tool for identification of Bordetella pertussis, B. holmesii, and B. parapertussis. Most United States public health laboratories (USPHLs) target IS481, present in 218-238 copies in the B. pertussis genome, and 32-65 copies in B. holmesii. CDC developed a multi-target PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis, and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with CDC. Spring and Fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, either in singleplex or multiplex assays. Seventy two percent and 79% of USPHLs could differentiate B. pertussis and B. holmesii in spring and fall, respectively; 68% and 72% could identify B. parapertussis. IS481 cycle threshold (Ct) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs who differentiated B. pertussis and B. holmesii, sensitivity and specificity was 96% and 95%, respectively, for the combined panels. The 2012 PEs demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to the previous survey.