MULTIPLEX PCR-BASED ASSAY FOR DETECTION OF BORDETELLA-PERTUSSIS IN NASOPHARYNGEAL SWAB SPECIMENS

A multiples PCR-based assay was developed for the detection of Bordete lla pertussis in nasopharyngeal swab specimens. The assay simultaneous ly amplified two separate DNA targets (153 and 203 bp) within a B. per tussis repetitive element and a 438-bp target within the beta-actin ge ne of human DNA (PCR amplification control). PCR products were detecte d by a sensitive and specific liquid hybridization gel retardation ass ay, A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture, Although 30 (6%) of the specime ns inhibited the amplification of the beta-actin target, in all 29 spe cimens studied, the inhibition disappeared on repeat testing or was ea sily overcome dth a 1:8 dilution or less of specimen digest. Of the 49 5 specimen pairs yielding a final evaluable result by the PCR-based as say, 19.0% were positive by the PCR-based assay, whereas 13.9% were po sitive by culture (P < 0.0001), After resolving the PCR-positive, cult ure-negative results by testing an additional aliquot from these speci mens by the multiples PCR-based assay, the PCR-based assay had a sensi tivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture, In comparison with patients with culture-confirmed pertussis, those with PCR-positive, c ulture-negative results were older and more likely to have had prolong ed cough, immunization with pertussis vaccine, or treatment with eryth romycin. This multiples PCR-based assay is substantially more sensitiv e than culture and identifies specimens that contain inhibitors of PCR .