Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens

Abstract

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the ␤-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the ␤-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity 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, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of Bordetella pertussis

“…PCR has repeatedly been shown to be more sensitive than culture and DFA for the detection of B. pertussis (4, 7-11, 13-15, 23, 24, 26, 27, 29). However, resolution of discrepant test results and assessment of the clinical performance of PCR have been carried out infrequently (13,26,29). The PCR test described in this study was shown to have excellent analytical sensitivity (Ͻ1 cell per PCR).…”

“…Accurate laboratory identification of B. pertussis infections remains problematic. Culture, long considered the "gold-standard" diagnostic method, is highly specific, but sensitivity can be affected by a number of factors, including patient age, immunization status, antibiotic treatment and duration of symptoms prior to specimen collection, and specimen transport conditions (28,29). The turnaround time for culture is typically 7 to 10 days for a negative specimen.…”

“…Notwithstanding this lack of standardization, PCR has consistently been shown to provide a more sensitive, specific, and rapid means of detecting B. pertussis infections than other methods (4, 7-11, 13-15, 23, 24, 26, 27, 29). While many studies have compared the positivity rates of culture and PCR tests for B. pertussis, relatively few have attempted to resolve the status of specimens that were positive by PCR alone and to determine the true clinical performance of the PCR test, particularly the specificity and positive predictive value (13,26,29).…”

Diagnosis of Human Metapneumovirus Infection in Immunosuppressed Lung Transplant Recipients and Children Evaluated for Pertussis

“…Suspensions of nasopharyngeal secretions were obtained from a collection maintained by the Pediatric Molec-ular Microbiology Laboratory at Children's Hospital of Pittsburgh (PA). The secretions were collected with Dacron swabs and suspended in 500 l of saline, and the suspensions were stored at Ϫ80°C as single-use aliquots (i.e., 100 l) until needed (25). The swab specimens had been obtained as part of routine care of pediatric patients who were evaluated for pertussis between February and May 2005.…”

Analysis with a Combination of Macrorestriction Endonucleases Reveals a High Degree of Polymorphism among Bordetella pertussis Isolates in Eastern France

“…One rapid screening method is based on the detection of calmodulin-dependent adenylate cyclase (AC)-hemolysin by an assay with an alginate swab specimen from the nasopharynx (7). Multitarget PCR-based assays (13,26) are sensitive and more efficient than culture, but nasopharyngeal sampling with Dacron swabs is required. No comparison of the results of the AC test and PCR-based diagnosis has been reported to date.…”