Two-Component Direct Fluorescent-Antibody Assay for Rapid Identification of Bacillus anthracis

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non–B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens

Prevalence and Sequence Variants of IS481 in Bordetella bronchiseptica: Implications for IS481-Based Detection of Bordetella pertussis

We report the prevalence in Bordetella bronchiseptica of IS481, a frequent target for diagnosis of Bordetella pertussis, as approximately 5%. However, PCR amplicons of the predicted size were detectable in 78% of IS481-negative strains. Our results suggest that PCR targeting IS481 may not be sufficiently specific for reliable identification of B. pertussis

Changes in Predominance and Diversity of Genomic Subtypes of Bordetella pertussis Isolated in the United States, 1935 to 1999

Pulsed-field gel electrophoresis (PFGE) of Bordetella pertussis chromosomal DNA fragments generated by XbaI restriction has been used to subtype isolates for epidemiologic studies. To better understand the natural history of pertussis, we determined the PFGE profiles of 1,333 strains isolated in the United States from 1935 to 1999. Results showed a shift in prevalent profiles from the earliest to the latest study periods. In addition, genetic diversity decreased over time, and prevalent profiles were more highly related to each other than to less common profiles. These results provide the foundation for investigating the impact of prevention strategies, including the use of the acellular vaccines, on the currently circulating B. pertussis population

Reproducibility of Bordetella pertussis Genomic DNA Fragments Generated by XbaI Restriction and Resolved by Pulsed-Field Gel Electrophoresis

The intra- and interlaboratory variabilities of the molecular size measurements of each DNA fragment contributing to three pulsed-field gel electrophoresis (PFGE) profiles were assessed, as were the reproducibilities of the entire PFGE profiles for three Bordetella pertussis strains. The major source of variability within a laboratory occurred between subcultures rather than within gels or between gels. Each PFGE profile was generated reproducibly and was objectively defined by the molecular sizes of its composite fragments. A strain or profile most suitable for use as an internal reference standard was identified

Analysis of Toxigenic Corynebacterium ulcerans Strains Revealing Potential for False-Negative Real-Time PCR Results▿

Diphtheria surveillance depends on the rapid and reliable recognition of the toxin gene in Corynebacterium diphtheriae. Real-time PCR is a rapid tool to confirm the presence of the diphtheria toxin gene (tox) in an isolate or specimen. We report that some toxigenic Corynebacterium ulcerans strains show atypical results in a real-time PCR for tox

Molecular Diagnosis of Bordetella pertussis Infection by Evaluation of Formalin-Fixed Tissue Specimens

Formalin-fixed lung or trachea tissue specimens from four infants and one adolescent who died of respiratory illness were tested for Bordetella pertussis by conventional and real-time PCR assays. B. pertussis was confirmed in all cases. PCR can be an invaluable retrospective diagnostic tool for evaluating archival tissues from patients with suspected fatal pertussis

Issues Associated with and Recommendations for Using PCR To Detect Outbreaks of Pertussis

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for Bordetella pertussis was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for B. pertussis during both outbreaks, which confirmed that B. pertussis was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that B. pertussis may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for B. pertussis. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease

Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer