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...

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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.

Bordetella pertussis, an obligate human pathogen and the agent of whooping cough, is a clonal species, despite the dynamic selection pressures imposed by host immunity and vaccine usage. Because the generation of variation is critical for species evolution, we employed a variety of approaches to examine features of B. pertussis genetic variation. We found a high level of conservation of gene content among 137 B. pertussis strains with different geographical, temporal, and epidemiological associations, using comparative genomic hybridization. The limited number of regions of difference were frequently located adjacent to copies of the insertion element IS481, which is present in high numbers in the B. pertussis chromosome. This repeated sequence appears to provide targets for homologous recombination, resulting in deletion of intervening sequences. Using subtractive hybridization, we searched for previously undetected genes in diverse clinical isolates but did not detect any new genes, indicating that gene acquisition is rare in B. pertussis. In contrast, we found evidence of altered gene order in the several strains that were examined and again found an association of IS481 with sites of rearrangement. Finally, we compared whole-genome expression profiles of different strains and found significant changes in transcript abundance, even in the same strain after as few as 12 laboratory passages. This combination of approaches provides a detailed picture of a pathogenic species with little gene loss or gain but with the capacity to generate variation by rearranging its chromosome and altering gene expression. These findings have broad implications for host adaptation by microbial pathogens.Bacterial pathogens face a dynamic array of selective pressures as they establish themselves within hosts, replicate, and are transmitted to new hosts. Upon arrival in a host, microbes may be forced to contend with innate immune defenses, scarcity of metabolic resources, competition from other bacteria for resources and colonization sites, and adaptive immunity, and then they must readapt to the outside environment during transmission to a new host. Individual hosts and pathogens adapt and respond to each other during the course of an infection, while evolving new offensive and defensive strategies on a population-wide basis. Pathogens must employ strategies to prevent their eradication by host immunity, which is constantly changing both within an individual host and within the host population. For an obligate pathogen to persist in a host population, its evolutionary process must be ongoing. This evolution can include gaining new functionality through horizontal transfer of genes, losing "antivirulence" genes, or altering the form or regulation of existing proteins through mutation in coding sequences or changes in promoters or operonic structures.The Bordetella genus of respiratory pathogens provides a rich model with which to explore questions of evolution of pathogenesis and host adaptation. The three species comprising the "c...

Numerous evaluations of the clinical sensitivity and specificity of PCR and serologic assays for Bordetella pertussis have been hampered by the low sensitivity of culture, the gold standard test, which leads to biased accuracy estimates. The bias can be reduced by using statistical approaches such as the composite reference standard (CRS) (e.g., positive if culture or serology positive; negative otherwise) or latent class analysis (LCA), an internal reference standard based on a statistical model. We illustrated the benefits of the CRS and LCA approaches by reanalyzing data from a 1995 to 1996 study of cough illness among 212 patients. The accuracy of PCR in this study was evaluated using three reference standards: culture, CRS, and LCA. Using specimens obtained 0 to 34 days after cough onset, estimates of the sensitivity of PCR obtained using CRS (47%) and LCA (34%) were lower than the culture-based estimate (62%). The CRS and LCA approaches, which utilized more than one diagnostic marker of pertussis, likely produced more accurate reference standards than culture alone. In general, the CRS approach is simple, with a well-defined disease status. LCA requires statistical modeling but incorporates more indicators of disease than CRS. When three or more indicators of pertussis are available, these approaches should be used in evaluations of pertussis diagnostic tests.Despite the availability of an effective pertussis vaccine since the mid-1940s, pertussis (whooping cough) remains endemic in the United States. In 2004, a total of 25,827 pertussis cases were reported to the Centers for Disease Control and Prevention (CDC) (24). Adolescents and adults accounted for the majority (67%) of reported cases. Laboratory diagnosis of pertussis is particularly difficult in these age groups, thereby limiting detection and control.For patients suspected of having pertussis, two types of clinical samples can be tested: a nasopharyngeal (NP) specimen for the isolation of Bordetella pertussis or for a PCR assay for B. pertussis DNA and a serum sample for the measurement of antibodies to B. pertussis antigens (11).B. pertussis isolation by microbial culture is the conventional gold standard for confirming pertussis (37, 60). Most studies have derived sensitivity and specificity estimates of PCR or serologic tests using culture results as the gold standard (37). Sensitivity is the proportion of the true diseased patients classified as positive, and specificity is the proportion of the true nondiseased patients classified as negative (62). However, culture is an insensitive test, because the organism is fastidious and often not recoverable from the nasopharynx more than 3 weeks after cough onset (37). Because culture has low sensitivity, it cannot be used to determine the true specificity of other or new pertussis tests. Both PCR assays for B. pertussis DNA and serologic assays for antibodies to B. pertussis antigens have not been standardized, and their sensitivity and specificity are incompletely defined (27,37,41,60).Consider a di...