Pertussis should be suspected in any infant death associated with marked leukocytosis, bronchopneumonia, or refractory pulmonary hypertension, particularly in children aged

To elucidate the potential role of the etiologic agent in recent increases of pertussis incidence in the United States, we studied the polymorphism in pertactin and pertussis toxin, which are Bordetella pertussis proteins important for pathogenesis and immunity. We sequenced regions of their genes (prn and ptx) in 152 B. pertussis strains isolated from 1935 through 1999 and identified 2 prn sequences: prn1 (old), observed continuously since 1935, and prn2 (new), not recognized until 1981 but seen in 97% of tested isolates in 1999. There were 3 ptx S1 subunit sequences: ptxS1D (old) was identified in 3 strains (1935 and 1939); ptxS1B (old) represented 87% of the strains recovered during 1935-1974; and ptxS1A (new) was the most prevalent during 1975-1987 and 1989-1999 (64% and 78%, respectively). Potential association between vaccination and the observed shift from old to new types requires further study. Our results provide the basis for prospectively monitoring for changes among circulating B. pertussis that might have epidemiologic relevance.

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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...

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycinbinding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.