Closed genome sequence of Clostridium botulinum type B1 strain isolated from an infant botulism case in the United States.

We present the closed genome sequence of the Clostridium botulinum BT-22100019 strain isolated from the stool specimen of an infant diagnosed with botulism. With 4.33-Mb genome size and 28.0% G + C content, the bont/B1 gene encoded for botulinum neurotoxin serotype B was found on a 262 kb plasmid arranged in a ha+ orfx - cluster.

Validation of a Lateral Flow Test for the Presumptive Identification of the Presence of Burkholderia mallei or Burkholderia pseudomallei in Environmental Samples.

We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader (OAR) for the rapid detection of B mallei or B pseudomallei in environmental (nonclinical) samples at 2 sites. The limit of detection, using reference strains B mallei strain ATCC 23344 and B pseudomallei strain ATCC 11668, was determined to be 103 to 104 CFU/mL. In different phases of the evaluation, inclusivity strains that included geographically diverse strains of B mallei (N = 13) and B pseudomallei (N = 22), geographically diverse phylogenetic near neighbor strains (N = 66), environmental background strains (N = 64), white powder samples (N = 26), and environmental filter extracts (N = 1 pooled sample from 10 filter extracts) were also tested. A total of 1,753 tests were performed, which included positive and negative controls. Visual and OAR results showed that the AMD test detected 92.3% of B mallei and 95.5% of B pseudomallei strains. Of the 66 near-neighbor strains tested, cross-reactivity was observed with only B stabilis 2008724195 and B thailandensis 2003015869. Overall, the specificity and sensitivity were 98.8% and 98.7%, respectively. The results of this evaluation support the use of the AMD test as a rapid, qualitative assay for the presumptive detection of B mallei and B pseudomallei in suspicious environmental samples such as white powders and aerosol samples by first responders and laboratory personnel.

Rapid Presumptive Identification of Burkholderia mallei and Burkholderia pseudomallei Clinical Isolates Using a Highly Specific Lateral Flow Assay.

We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader for the rapid identification of culture isolates of B mallei or B pseudomallei to support clinical diagnosis for response and triage during a mass casualty event, such as a biological attack. The study was conducted at 2 sites to assess the performance of the AMD test. The sensitivity, specificity, and reproducibility of the assay was determined using 5 replicates of 35 inclusivity strains and 64 clinical background strains. A total of 520 tests were performed, which included both positive and negative controls. Results obtained visually and with the Omni Array Reader showed a sensitivity of 92.3% for B mallei and 95.6% for B pseudomallei; no cross-reactivity was observed with any of the 64 clinical background organisms. The results from this study indicate that the AMD test for the presumptive identification of B mallei and B pseudomallei isolates to support clinical diagnosis is highly robust, specific, and sensitive. This evaluation supports the use of this test as a rapid, qualitative assay for the presumptive identification of B mallei and B pseudomallei in a clinical setting.

Whole-Genome Assessment of Clinical Acinetobacter baumannii Isolates Uncovers Potentially Novel Factors Influencing Carbapenem Resistance.

Carbapenems-one of the important last-line antibiotics for the treatment of gram-negative infections-are becoming ineffective for treating Acinetobacter baumannii infections. Studies have identified multiple genes (and mechanisms) responsible for carbapenem resistance. In some A. baumannii strains, the presence/absence of putative resistance genes is not consistent with their resistance phenotype-indicating the genomic factors underlying carbapenem resistance in A. baumannii are not fully understood. Here, we describe a large-scale whole-genome genotype-phenotype association study with 349 A. baumannii isolates that extends beyond the presence/absence of individual antimicrobial resistance genes and includes the genomic positions and pairwise interactions of genes. Ten known resistance genes exhibited statistically significant associations with resistance to imipenem, a type of carbapenem: blaOXA-23, qacEdelta1, sul1, mphE, msrE, ant(3")-II, aacC1, yafP, aphA6, and xerD. A review of the strains without any of these 10 genes uncovered a clade of isolates with diverse imipenem resistance phenotypes. Finer resolution evaluation of this clade revealed the presence of a 38.6 kbp conserved chromosomal region found exclusively in imipenem-susceptible isolates. This region appears to host several HTH-type DNA binding transcriptional regulators and transporter genes. Imipenem-susceptible isolates from this clade also carried two mutually exclusive plasmids that contain genes previously known to be specific to imipenem-susceptible isolates. Our analysis demonstrates the utility of using whole genomes for genotype-phenotype correlations in the context of antibiotic resistance and provides several new hypotheses for future research.

Evaluation of an Electrochemiluminescence Assay for the Rapid Detection of Abrin Toxin.

In this article, we detail a comprehensive laboratory evaluation of an immunoassay for the rapid detection of abrin using the Meso Scale Diagnostics Sector PR2 Model 1800. For the assay evaluation, we used inclusivity and exclusivity panels comprised of extracts of 11 Abrus precatorius cultivars and 35 near-neighbor plants, 65 lectins, 26 white powders, 11 closely related toxins and proteins, and a pool of 30 BioWatch filter extracts. The results show that the Meso Scale Diagnostics abrin detection assay exhibits good sensitivity and specificity with a limit of detection of 4 ng/mL. However, the dynamic range of the assay for the quantitation of abrin was limited. We observed a hook effect at higher abrin concentrations, which can lead to potential false negative results. A modification of the assay protocol that incorporates extra wash steps can decrease the hook effect and the potential for false negative results.

Comprehensive Laboratory Evaluation of a Specific Lateral Flow Assay for the Presumptive Identification of Francisella tularensis in Suspicious White Powders and Aerosol Samples.

We conducted a comprehensive, multi-phase laboratory evaluation of the Tularemia BioThreat Alert® (BTA) test, a lateral flow assay (LFA) for the rapid detection of Francisella tularensis. The study, conducted at 2 sites, evaluated the limit of detection (LOD) of this assay using the virulent SchuS4 strain and the avirulent LVS strain of F. tularensis. In 6-phase evaluation (linear dynamic range and reproducibility, inclusivity, near-neighbor, environmental background, white powder, and environmental filter extract), 13 diverse strains of F. tularensis, 8 Francisella near neighbors, 61 environmental background organisms, 26 white powders, and a pooled aerosol extract were tested. In the 937 tests performed, the Tularemia BTA demonstrated an LOD of 107 to 108 cfu/mL, with a sensitivity of 100.00%, specificity of 98.08%, and accuracy of 98.84%. These performance data are important for accurate interpretation of qualitative results arising from screening suspicious white powders in the field.

Comprehensive Laboratory Evaluation of a Lateral Flow Assay for the Detection of Yersinia pestis.

We conducted a comprehensive, multiphase laboratory evaluation of the Plague BioThreat Alert® (BTA) test, a lateral flow immunoassay (LFA), for the rapid detection of Yersinia pestis. The study was conducted in 7 phases at 2 sites to assess the performance of the LFA. The limit of detection (LOD) was determined using both a virulent and avirulent strain of Y. pestis, CO99-3015 (105 CFU/ml) and A1122 (104 CFU/ml), respectively. In the other phases, 18 Y. pestis strains, 20 phylogenetic near-neighbor strains, 61 environmental background microorganisms, 26 white powders, and a pooled aerosol sample were also tested. A total of 1,110 LFA test results were obtained, and their analysis indicates that this LFA had a sensitivity of 97.65% and specificity of 96.57%. These performance data are important for accurate interpretation of qualitative results arising from testing suspicious white powders and aerosol samples in the field. Any positive specimen in this assay is considered presumptive positive and should be referred to the Centers for Disease Control and Prevention Laboratory Response Network for additional testing, confirmation, and characterization for an appropriate public health response.

Rapid Presumptive Identification of Bacillus anthracis Isolates Using the Tetracore RedLine Alert™ Test.

A comprehensive laboratory evaluation of the Tetracore RedLine Alert test, a lateral flow immunoassay (LFA) for the rapid presumptive identification of Bacillus anthracis, was conducted at 2 different test sites. The study evaluated the sensitivity of this assay using 16 diverse strains of B. anthracis grown on sheep blood agar (SBA) plates. In addition, 83 clinically relevant microorganisms were tested to assess the specificity of the RedLine Alert test. The results indicated that the RedLine Alert test for the presumptive identification of B. anthracis is highly robust, specific, and sensitive. RedLine Alert is a rapid test that has applicability for use in a clinical setting for ruling-in or ruling-out nonhemolytic colonies of Bacillus spp. grown on SBA medium as presumptive isolates of B. anthracis.

Closed Genome Sequence of Clostridium botulinum Strain CFSAN064329 (62A).

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies. We report here the closed genome sequence of Clostridium botulinum strain CFSAN064329 (62A).

Use of quantitative flow cytometry to measure ex vivo immunostimulant activity of echinacea: the case for polysaccharides.

INTRODUCTION: When directly exposed to various echinacea fractions, human leukocytes ex vivo are strongly stimulated to proliferate and to produce immunostimulation and inflammatory cytokines. A comparison of fractions containing lipoidal small molecules and high-molecular-weight water-soluble polysaccharides indicates that the latter are substantially more potent as immunostimulants. Echinacea purpurea (L.) Moench, E. angustifolia DC, and E. pallida (Nutt.), Nutt. extracts, and each plant part contain significantly potent constituents. Flow cytometric techniques were utilized. OBJECTIVES: This study was undertaken to determine whether flow cytometry could measure immunostimulant activity present in echinacea and, if so, which species produced more activity, which plant part was the most active, and whether the organic soluble or the aqueous extractables were more active. Ex vivo human clinical material was employed. DESIGN: Echinacea extracts were analyzed using flow cytometric techniques. The immunostimulation assays were measured in triplicate. METHODS: Samples dissolved in dimethyl sulfoxide (DMSO) were added to 200 microL of heparinized blood mixed with 50 muL of phosphate buffer, vortexed, and incubated to allow adequate time for immune-cell stimulation. Fifty (50) microL of the stimulated blood samples were added to each of a reagent cocktail consisting of 20 microL of CD4FITC/CD69PE/CD3PerCP expressed on the helper/inducer T-lymphocyte subset; CD8FITC/CD69/PE/ CD3PerCP expressed on the human suppresser/cytotoxic T-lymphocytes and on a subset of natural killer lymphocytes; CD19FITC/CD69PE/CD45PerCP expressed on B-lymphocytes; or CD56FITC/CD69PE/CD45PerCP expressed on NK lymphocytes. Four hundred and fifty (450) microL of 1 X FACS lysing solution was added and incubated in the dark (rt, 30 minutes) and then subjected to flow cytometric analysis. All reported readings are the average of several determinations. Positive controls consisted of phorbol myristyl acetate (PMA) (50 ng/mL), phytohemagglutinin (10 microg/mL), CD2/CD2R (positive activation control)(5 microL/250 muL of reaction), and negative controls consisted of dimethyl sulfoxide (2% in RPMI-1640), RPMI-1640 medium, and cyclosporin A (10 microg/mL). RESULTS: The main immunostimulatory activity of echinacea resides in the water-soluble materials rather than the lipoidal small molecules. E. purpurea, E. Pallida, and E. angustifolia leaves, stems, flowering tops, and roots all produce substantial immunostimulatory activity. CONCLUSIONS: The use of flow cytometry demonstrates a link between the polysaccharides in echinacea and the biologic immunostimulatory effect that has therapeutic relevance, and strong evidence for this immunostimulant property is presented.