Real-Time PCR Assay Targeting IS 481 of Bordetella pertussis and Molecular Basis for Detecting Bordetella holmesii

Abstract

Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported.Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.Bordetella pertussis is the causative agent of whooping cough, an infectious disease that occurs worldwide with a high prevalence among young, unvaccinated infants (2,3,6,19) and is recently resurgent in highly vaccinated populations. Related species, including B. parapertussis, B. bronchiseptica, and the more recently described B. holmesii (27), may also cause a pertussis-like syndrome in humans. Laboratory diagnosis of pertussis is traditionally based primarily on culture, which is highly specific but is maximally sensitive only in the initial phases of disease (10,14). Furthermore, culturing depends on specimen quality and laboratory expertise and requires special media, extended incubation periods of 7 days or more, and confirmation by biochemical or antibody reagent tests. Diagnostic serology can be highly sensitive and more rapid than culture (14), but no serologic assay has been approved for diagnostic use in the United States because no diagnostic criterion has been widely accepted and no method has been validated between laboratories.Thus, there is a need for more rapid and sensitive diagnostic methods that have high positive predictive value, especially in the early stages of disease. As is the case for other fastidious organisms, PCR offers an attractive alternative for detecting B. pertussis and B. parapertussis in clinical specimens (1,4,5,7,15,17,21,26). Previously evaluated B. pertussis target regions include insertion sequences (IS), repeat elements, the pertussis toxin promoter region, the adenylate cyclase gene, and the porin gene. IS481 is present in the B. pertussis genome at 80 (22) to 100 (5, 18) copies, and PCR assays targeting IS481 have been evaluated for sensitivity and specificity in several laboratories over the last few years. Therefore, an internal region of IS481 was selected as the B. pertussis target (18) while a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) duplex PCR for B. pertussis and B. parapertussis was being developed. The previously described (5) forward primer BP-1 (5Ј GAT TCA ATA GGT TGT ATG CAT GGT T 3Ј and the slightly modified reverse primer BP-2 (5Ј TTC AGG CAC ACA AAC TTG ATG GGC G 3Ј), corresponding to bp 12 to 36 and 192 to 167 (GenBank M22031) of IS481, respectively, were used for amplification. A pair of fluorescence-labeled hybridization probes, BP-HP-3 (5Ј TCG CCA ACC CCC CAG TTC ACT CA-FAM 3Ј) and BP-HP-4 (5Ј LC red 640-AGC CCG GCC GGA TGA ACA CCC-3Ј-phosphate), corresponding to bp 66 to 88 and 92 to 112 (GenBank M22031) of IS481, respecti...

Laboratory diagnosis of pertussis infections: the role of PCR and serology

“…Only B. pertussis isolates gave bands of the expected size in the ptxA-Pr and IS481 assays. This is despite the reported presence of IS481-like elements in both B. holmesii (Reischl et al, 2001) and B. bronchiseptica (Gladbach et al, 2002). Explanations for the lack cross-reactivity with these two species using our IS481 PCR assay are: different sensitivities of the assays, the low and variable copy number of these elements in B. holmesii and B. bronchiseptica or the difference in the primer (probe) combination used and possible sequence variation in the target regions of these oligonucleotides.…”

“…A PCR assay may be thought to be specific for an organism when it is designed, but subsequently it may be found that its target gene is present in other closely related, or even unrelated, organisms. Although in this study no amplification was seen with purified DNA from B. holmesii or B. bronchiseptica, the presence of IS481-like sequences in these two additional Bordetella species potentially compromises the use of such assays in establishing the specific presence of B. pertussis (Reischl et al, 2001;Gladbach et al, 2002). However, it appears that the greater sensitivity of IS481 assays (due to the presence of multiple copies) is a factor in their continued use by several laboratories.…”

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].…”

Direct molecular typing of Bordetella pertussis from clinical specimens submitted for diagnostic quantitative (real-time) PCR

“…The first target, within the pertussis toxin S1 promoter (ptxP; previously denoted ptxA-pr), is specific for B. pertussis and is present at 1 copy per bacterial genome. The second target, the insertion sequence IS481, occurs in multiple copies in B. pertussis strains, but may also be present in strains of Bordetella parapertussis, Bordetella bronchiseptica and Bordetella holmesii (Muyldermans et al, 2005;Register & Sanden, 2006;Reischl et al, 2001;Fry et al, 2009). Despite the age restriction on the Health Protection Agency's diagnostic qPCR service, the number of pertussis cases confirmed by qPCR is increasing each year.…”