Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay

<p>Abstract</p> <p>Background</p> <p>A low genetic diversity in <it>Francisella tularensis </it>has been documented. Current DNA based genotyping methods for typing <it>F. tularensis </it>offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.</p> <p>Results</p> <p>We have generated a high-resolution phylogenetic tree from 40 <it>Francisella </it>isolates, including 13 <it>F. tularensis </it>subspecies <it>holarctica </it>(type B) strains, 26 <it>F. tularensis </it>subsp. <it>tularensis </it>(type A) strains and a single <it>F. novicida </it>strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^®resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of <it>F. tularensis </it>subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional <it>F. tularensis </it>strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).</p> <p>Conclusion</p> <p>Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of <it>F. tularensis </it>subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of <it>F. tularensis </it>to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.</p

Avian cholera in a southern giant petrel (Macronectes giganteus) from Antarctica

A southern giant petrel (Macronectes giganteus) was found dead at Potter Peninsula, King George Island, South Shetland, Antarctica. The adult male was discovered approximately 48 hr after death. Macroscopic and microscopic lesions were compatible with avian cholera and the bacterium Pasteurella multocida subsp. gallicida, serotype A1 was isolated from lung, heart, liver, pericardial sac, and air sacs. In addition, Escherichia coli was isolated from pericardial sac and air sacs. This is the first known report of avian cholera in a southern giant petrel in Antarctica.Facultad de Ciencias Veterinaria

Ellagic Acid Derivatives from Rubus ulmifolius Inhibit Staphylococcus aureus Biofilm Formation and Improve Response to Antibiotics

Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections.This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50-200 µg/mL, which were well below the concentrations required to limit bacterial growth (530-1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects.These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections

Subsequent Surgery After Revision Anterior Cruciate Ligament Reconstruction: Rates and Risk Factors From a Multicenter Cohort

BACKGROUND: While revision anterior cruciate ligament reconstruction (ACLR) can be performed to restore knee stability and improve patient activity levels, outcomes after this surgery are reported to be inferior to those after primary ACLR. Further reoperations after revision ACLR can have an even more profound effect on patient satisfaction and outcomes. However, there is a current lack of information regarding the rate and risk factors for subsequent surgery after revision ACLR. PURPOSE: To report the rate of reoperations, procedures performed, and risk factors for a reoperation 2 years after revision ACLR. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A total of 1205 patients who underwent revision ACLR were enrolled in the Multicenter ACL Revision Study (MARS) between 2006 and 2011, composing the prospective cohort. Two-year questionnaire follow-up was obtained for 989 patients (82%), while telephone follow-up was obtained for 1112 patients (92%). If a patient reported having undergone subsequent surgery, operative reports detailing the subsequent procedure(s) were obtained and categorized. Multivariate regression analysis was performed to determine independent risk factors for a reoperation. RESULTS: Of the 1112 patients included in the analysis, 122 patients (11%) underwent a total of 172 subsequent procedures on the ipsilateral knee at 2-year follow-up. Of the reoperations, 27% were meniscal procedures (69% meniscectomy, 26% repair), 19% were subsequent revision ACLR, 17% were cartilage procedures (61% chondroplasty, 17% microfracture, 13% mosaicplasty), 11% were hardware removal, and 9% were procedures for arthrofibrosis. Multivariate analysis revealed that patients aged <20 years had twice the odds of patients aged 20 to 29 years to undergo a reoperation. The use of an allograft at the time of revision ACLR (odds ratio [OR], 1.79; P = .007) was a significant predictor for reoperations at 2 years, while staged revision (bone grafting of tunnels before revision ACLR) (OR, 1.93; P = .052) did not reach significance. Patients with grade 4 cartilage damage seen during revision ACLR were 78% less likely to undergo subsequent operations within 2 years. Sex, body mass index, smoking history, Marx activity score, technique for femoral tunnel placement, and meniscal tearing or meniscal treatment at the time of revision ACLR showed no significant effect on the reoperation rate. CONCLUSION: There was a significant reoperation rate after revision ACLR at 2 years (11%), with meniscal procedures most commonly involved. Independent risk factors for subsequent surgery on the ipsilateral knee included age <20 years and the use of allograft tissue at the time of revision ACLR

ENVIRONMENTAL CONTAMINANTS ASSOCIATED WITH A SWINE CONCENTRATED ANIMAL FEEDING OPERATION AND IMPLICATIONS FOR MCMURTREY NATIONAL WILDLIFE REFUGE

Waste generated by concentrated animal feeding operations (CAFOs) may contain a variety of contaminants including nutrients, pathogens, trace elements, antibiotics, and hormones. In 2000, the U.S. Fish and Wildlife Service began to characterize CAFO contaminants in lagoons, canals, and created wetlands operated by Hastings Pork, a large swine CAFO adjacent to McMurtrey National Wildlife Refuge (McMurtrey) in Clay County, Nebraska. The created wetlands were designed to attract waterfowl; therefore, the primary purpose of this research was to evaluate whether migratory waterfowl were likely exposed to CAFO contaminants. A secondary research objective was to determine if created wetland water was suitable as a supplementary water source for McMurtrey. Wetlands created from swine wastewater effluent had 5-50 fold greater concentrations of phosphorus, ammonia, and total nitrogen and 2-3 fold greater salinity compared to control sites. Cyanobacteria (Microcystis spp.) were abundant in the created wetlands and microcystin toxins were detected in concentrated water samples. Tetracycline, macrolide, and diterpene antibiotics were detected in lagoon and canal sediment and water samples; however, in the created wetlands only oxytetracycline was detected (once in sediment at 41 nanograms per gram). Concentrations of 17-β estradiol and testosterone in CAFO wastewater (n=4) exceeded toxicity thresholds for aquatic life. Fecal coliform and streptococci counts in water (n=38) generally exhibited a decreasing gradient with lagoons \u3e canals \u3e created wetlands \u3e McMurtrey. Bacteria (Salmonella spp. and Yersinia enterocolitica) were recovered in the created wetlands but not McMurtrey. Created wetland invertebrate communities were dominated by chironomid species and had lower taxa diversity when compared to McMurtrey. Eutrophication of created wetlands may represent the greatest health threat to waterfowl by creating an environment conducive to cyanobacteria blooms and outbreaks of avian botulism and avian cholera. Trace elements from swine waste will likely continue to accumulate in the created wetlands over time, leading to an increased risk of exposure to wetland biota. Research is ongoing and includes use of sentinel mallards (Anas platyrhynchos) to further evaluate the need to decrease concentrations of CAFO contaminants in swine wastewater before it is used to create waterfowl habitat