Evaluation of Real-Time PCR for Detection of and Discrimination between Bordetella pertussis , Bordetella parapertussis , and Bordetella holmesii for Clinical Diagnosis

Abstract

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

A multiplex PCR for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in clinical specimens

“…Positive results should be followed by species-discriminating tests [33,34] to achieve diagnostic levels of specificity [28,31]. This test will not amplify sequence from B. parapertussis [33], and should not amplify from other known Bordetella species based on the apparent absence of the IS481 element outside of B. pertussis and B. holmesii [28,33].…”

“…This test will not amplify sequence from B. parapertussis [33], and should not amplify from other known Bordetella species based on the apparent absence of the IS481 element outside of B. pertussis and B. holmesii [28,33].…”

Septic arthritis caused by Bordetella holmesii in an adolescent with chronic haemolytic anaemia

“…In our case, the positivity of the IS481 real-time PCR indicates the usefulness of this method to search for B. holmesii, a species that like B. pertussis harbours IS481 (Templeton et al, 2003). However, molecular techniques like 16S rRNA gene-based PCR could be useful, as in many cases, the culture result may be falsely negative for various reasons, the most frequent being patients starting antibiotic treatment before adequate samples are taken.…”

“…Of interest, the real-time PCR detection of B. pertussis/B. holmesii species based on IS481 detection performed retrospectively on DNA extract from the joint fluid as described elsewhere (Templeton et al, 2003;Bidet et al, 2008) was positive, confirming the presence of the pathogen in the knee joint. The blood culture was negative.…”

Implementation and Assessment of the Use of Real-Time PCR in Routine Diagnosis for Bordetella pertussis Detection in Brazil

“…Several PCR protocols have been developed in recent years for different targets in the genome of B. pertussis like an insertion sequence IS481, pertussis toxin gene promoter (ptxA-Pr), pertussis toxin S1 subunit (ptxS1), porin gene, pertactin filaments hemagglutinin gene and adenylate cyclase (14,15,20,21). Many laboratories use the insertion sequence IS481 in PCR assays to determine the presence of B. pertussis DNA.…”