Description of Pertussis PCR Assays Used By Laboratories in Washington State During a Pertussis Epidemic

BACKGROUND: In 2012, Washington (WA) observed the highest number of pertussis cases reported in over 70 years with 4,783 cases.  High rates were reported in infants and in adolescents with high vaccination coverage. Among lab-confirmed cases (n=3,481), 95% were detected by PCR alone. Though highly sensitive, pertussis PCR assays are not standardized and pseudo-outbreaks due to contamination and Bordetella holmesii have been documented. We surveyed laboratories in WA to determine the PCR practices and assay types used during the epidemic.

METHODS: Using data from the WA Department of Health’s Office of Laboratory Quality Assurance, we identified 47 laboratories in WA which are regulated and perform primarily non-waived testing. To assess pertussis PCR practices, an online survey was emailed to lab supervisors and microbiology leads to determine the type of assays and probes, DNA extraction methods, and interpretation criteria being used.  Labs not performing onsite testing were asked to identify their reference laboratory. Follow-up by phone was conducted with non-responding and out-of-state reference labs.

RESULTS: Of the 47 WA labs identified, 37 (79%) responded to the online survey and the remaining 10 completed the survey by phone.  The majority (87%) forward pertussis nasopharyngeal swabs to reference labs for PCR testing.  Five labs (11%) conduct PCR onsite and one lab (2%) doesn’t test for pertussis.  Four out-of-state reference labs were identified from WA lab respondents and were subsequently surveyed. All nine labs performing onsite testing use real-time PCR and the majority (78%) use in-house assays. Seven of these labs report using PCR assays targeting IS481, present in multiple copies of B. pertussis and holmesii, and IS1001 for B. parapertussis.  One of these labs (a large hospital lab in WA) also uses the pertussis toxin promoter, and RecA targets for B. holmesii.  Two labs target IS481 only. Extraction methods, the number of amplification cycles and interpretation criteria varied.

CONCLUSIONS:  There is a lack of standardization for pertussis PCR testing in WA and only one lab could identify B. holmesii.  Improved understanding of pertussis PCR assays used by testing labs may help avoid pitfalls associated with PCR and improve confidence in the determination of the etiology of outbreaks.  Our results highlight the need for multi-target PCR testing capacity at our state public health lab in order to rapidly rule out pseudo-outbreaks or other Bordetella species that can cause pertussis-like illness during localized outbreaks and to help guide public health interventions.