External quality assurance scheme on PCR for Bordetella pertussis, 2012

Twenty-one laboratories from 21 EU/EEA countries participated in the first external quality assurance scheme for Bordetella pertussis PCR by ECDC on behalf of the EUpert-labnet network. The panel included dilutions of purified genomic DNA from B. pertussis at three concentrations designated 'high', 'medium' and 'low'. Duplicate samples of the B. pertussis 'medium' dilution were included to test the reproducibility of the PCR assays. Genomic DNA from other Bordetella species - B. parapertussis, B. holmesii, B. bronchiseptica - were included in the panel, together with Haemophilus influenzae. Two 'blank' samples, i.e. with no added DNA, were also included to check for potential contamination. Of 22 datasets (one laboratory submitted two datasets) all (100%) reported the 'high' concentration as positive for B. pertussis and 21/22 also reported the 'medium' concentration duplicate samples as B. pertussis positive. Only 15 out of 22 reported the 'low' concentration as positive for B. pertussis or Bordetella spp. Real-time Bordetella PCR assays (both in-house and commercial) demonstrated greater sensitivity than conventional PCR assays as demonstrated by 85% (11/13) of those using qPCR reporting the 'low' concentration positive for B. pertussis or Bordetella spp. compared with 57% (4/7) of those using conventional PCR. None of the laboratories reported Bordetella DNA in the two blank samples, demonstrating good molecular laboratory practice. The most common targets for B. pertussis and B. parapertussis were IS481 and IS1001, respectively. However, these targets are not completely specific for these species. Therefore IS481 positive-only results should be reported as probable B. pertussis (B. pertussis / B. holmesii / B. bronchiseptica) and IS1001 positive-only results should be reported as probable B. parapertussis (B. parapertussis / B. bronchiseptica). Only laboratories using specific B. holmesii assays (e.g. targeting recA) could correctly differentiate this species from other Bordetella species including B. pertussis. Several commercial kits are available for the detection of B. pertussis and/or B. parapertussis; however, care should be taken in the interpretation of these results as indicated above. An internal process control to check for the presence of PCR inhibitors is recommended to avoid false-negative reporting. This report presents the results of the first external quality assurance (EQA) scheme for Bordetella pertussis PCR funded by the European Centre for Disease Prevention and Control (ECDC). The EQA study was conducted between February and March 2012.