VALIDATION OF NESTED BORDETELLA PCR IN PERTUSSIS-VACCINE TRIAL

A nested PCR, using a 239-bp sequence in the pertussis toxin promoter region, was developed and evaluated, The assay differentiates Bordetel la pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by restriction enzyme analysis of the amplified fragments, The diagnos tic performance of the PCR was evaluated in a Swedish pertussis vaccin e efficacy trial which took place from 1992 to 1995, including study c hildren and household members and using culture and serology for labor atory confirmation of suspected cases, In total 2,421 nasopharyngeal a spirates were analyzed, The total diagnostic sensitivity for B, pertus sis was 90.2% (194 of 215), During the study period samples were proce ssed with and without the cation-exchange resin Chelex, The PCR diagno stic sensitivity for B, pertussis among the Chelex-treated aspirates w as 94.9% (75 of 79), and that for B, pertussis among 124 aspirates in a consecutive non-Chelex-treated material was 89.5% (111 of 124). Afte r Chelex treatment of the 13 PCR-negative samples, an additional six b ecame PCR positive, giving a final sensitivity of 94.3%, In addition, PCR was positive for B. pertussis with 57 of 1,744 samples negative by culture but with available serological data, The specificity of PCR w ith these samples was supported by a significant increase in antibody levels between acute and convalescent sera in 45 cases and by epidemio logical or clinical data in all but two of the remaining cases, PCR wa s also positive for B, pertussis with 26 of 415 aspirates from episode s lacking serology. The diagnostic sensitivity of PCR for B. parapertu ssis was 74.0% (37 of 50), There were an additional seven culture-nega tive B, parapertussis PCR findings, six from cases with significant an tibody increases against filamentous hemagglutinin only and one from a case lacking serology, There were no samples positive for B, bronchis eptica. In conclusion, PCR detection of B, pertussis and/or B, paraper tussis enabled us to identify 90 positive nasopharyngeal aspirates, in addition to the 262 culture-positive samples (an increase of 34%), Re lating these cases to serology and clinical data indicated a PCR speci ficity approaching 100%.