[Show abstract][Hide abstract] ABSTRACT: Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96- 3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed.
[Show abstract][Hide abstract] ABSTRACT: Tularemia in the United States was examined by reviewing 106 Francisella tularensis isolates, mostly from Nebraska, collected during 1998-2012: 48% of Nebraska cases were cat-associated; 7/8 human cases were caused by subtype A.I. A vaccine is needed to reduce feline-associated tularemia, and cat owners should protect against bites/scratches and limit their pet's outdoor access.
[Show abstract][Hide abstract] ABSTRACT: This study investigated the diagnostic accuracy of whole slide imaging (WSI) in breast needle biopsy diagnosis in comparison with standard light microscopy (LM). The study examined the effects of image capture magnification and computer monitor quality on diagnostic concordance of WSI and LM.
Four pathologists rendered diagnoses using WSI to examine 85 breast biopsies (92 parts; 786 slides) consisting of benign and malignant cases. Each WSI case was evaluated using images captured at either 20x or 40x magnifications and viewed using a DICOM grade, color-calibrated monitor or a standard, desktop LCD monitor. For each combination, the WSI result was compared to the original, LM diagnosis. The overall concordance rate observed between WSI and LM was 97.1% (95% CI:94.3%-98.5%). After a washout period, all cases were reviewed a second time by each pathologist after using LM, and the second LM diagnosis was compared to the WSI diagnosis rendered by the same pathologist. Intraobserver concordance between WSI and LM was 95.4% (95% CI:92.2%- 97.4%). The second LM diagnoses were also compared to the original LM diagnoses, and the observed interobserver LM concordance rate was 97.3% (95% CI:93.1% -99.0%).
The study data demonstrated that breast needle biopsy diagnoses rendered by WSI were equivalent to diagnoses rendered by LM. No diagnostic differences were detected between the underlying viewing system parameters of monitor quality and image capture resolution. The results of this study demonstrated that WSI can be effectively utilized in subspecialty diagnostic cases where a minimum amount of tissue is available.
Human pathology 08/2014; 45(8). DOI:10.1016/j.humpath.2014.04.007 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human butyrylcholinesterase (BChE) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, recombinant BChE (rBChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 µM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 µM) for 1.5 h at 25˚C. However, rBChE tetramerization was inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellular machinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15 to 50 consecutive proline residues.
[Show abstract][Hide abstract] ABSTRACT: This research investigated the use of SNOMED CT to represent diagnostic tissue morphologies and notable tissue architectures typically found within a pathologist's microscopic examination report to identify gaps in expressivity of SNOMED CT for use in anatomic pathology.
24 breast biopsy cases were reviewed by two board certified surgical pathologists who independently described the diagnostically important tissue architectures and diagnostic morphologies observed by microscopic examination. In addition, diagnostic comments and details were extracted from the original diagnostic pathology report. 95 unique clinical statements were extracted from 13 malignant and 11 benign breast needle biopsy cases.
75% of the inventoried diagnostic terms and statements could be represented by valid SNOMED CT expressions. The expressions included one pre-coordinated expression and 73 post-coordinated expressions. No valid SNOMED CT expressions could be identified or developed to unambiguously assert the meaning of 21 statements (ie, 25% of inventoried clinical statements). Evaluation of the findings indicated that SNOMED CT lacked sufficient definitional expressions or the SNOMED CT concept model prohibited use of certain defined concepts needed to describe the numerous, diagnostically important tissue architectures and morphologic changes found within a surgical pathology microscopic examination.
Because information gathered during microscopic histopathology examination provides the basis of pathology diagnoses, additional concept definitions for tissue morphometries and modifications to the SNOMED CT concept model are needed and suggested to represent detailed histopathologic findings in computable fashion for purposes of patient information exchange and research.
UNMC Institutional Review Board ID# 342-11-EP.
Journal of the American Medical Informatics Association 05/2014; 21(5). DOI:10.1136/amiajnl-2013-002456 · 3.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute respiratory infections are a significant concern throughout the world, contributing to 4.25 million deaths annually. One such pathogen, H5N1 avian influenza, has the potential to become the next pandemic threat. While currently not transmitted human-to-human, recent cases of H5N1 have been shown to result in a 60% fatality rate and an increasing resistance to antiviral treatments. However, because of the rapid mutation rate of RNA viruses like influenza, it is likely that human-to-human transmission is imminent, motivating the need for efficacious vaccines against pandemic influenzas.
Polyanhydride nanovaccines have been shown to be a versatile platform for the delivery of subunit vaccines. Nanoparticles composed of sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy) hexane (CPH), and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), and their copolymers have been shown to sustain the release of encapsulated proteins while enhancing internalization by antigen presenting cells. Polyanhydride nanovaccines have also been shown to have immunomodulatory capabilities inducing both high avidity antibody titers as well as cell-mediated immune responses.
Previously, we have demonstrated that polyanhydride nanoparticles are capable of stably releasing recombinant H5 hemagglutinin, an antigen for H5N1 influenza. In this work, we characterized the in vivo immune responses generated by polyanhydride-based subunit nanovaccines. Polyanhydride nanovaccine formulations encapsulating rH5 were found to elicit high antigen-specific antibody titers as well as virus-neutralizing antibody titers. Likewise, enhanced T cell memory responses were observed at 63 days post immunization. Finally, the efficacy of a nanovaccine formulation was evaluated using a live viral challenge. All these studies together indicate that polyanhydride nanovaccines offer a patient-friendly and efficacious platform for next generation influenza A vaccines.
[Show abstract][Hide abstract] ABSTRACT: Patient: 75-year-old Caucasian male. Chief Complaint: Frequent diarrhea. History of Present Illness: The patient had a history of Crohn's disease, initially diagnosed in 1976 with a positive test for toxigenic Clostridium difficile (C. difficile) in August 2011, successfully treated with methronidazole (500 mg PO bid ×10 d). There was a concern that diarrhea may be a result of C. difficile recurrence. Past Medical History: The patient had an extensive medical history, including eosinophilia (8%, normal 0%-7%) in October 2011, gastroesophageal reflux disease, anemia secondary to Crohn's disease, coronary artery disease, hypertension, osteoarthritis, and Type II diabetes mellitus. Additionally, the patient has severe Crohn's disease which involves the terminal ileum and colon, and has undergone multiple small bowel resections as well as a colectomy. Medications included immunomodulatory therapy with balsalazide (Colazal), azathioprine (Imuran), infliximab (Remicade), ranitidine, and prednisone for both Crohn's disease and osteoarthritis. Previous biopsies of the small intestine and colon (before the current presentation) indicated changes consistent with Crohn's disease. Travel History: The patient was originally from Louisiana, and briefly lived in Las Vegas, NV. For approximately 40 years, he has lived in western Iowa. The patient did not have a history of foreign travel nor had he recently traveled outside of Iowa or Nebraska. He drives a bus for a local business. Principle Laboratory Findings: Loose brown stool was collected in December 2011 and submitted for laboratory testing for toxigenic C. difficile and cultured for enteric pathogens. An ova and parasites exam was not ordered at that time. Salmonella, Shigella, and Campylobacter species, as well as Shiga toxin producing-Escherichia coli, were not detected; the toxigenic C. difficile assay was negative. A laboratory technologist noted the presence of small trails of displaced bacteria on the blood agar plate from the original stool culture (Image 1). From this, a full ova and parasites exam was performed on the stool specimen.
Laboratory Medicine 10/2013; 44(4):339-343. DOI:10.1309/LMI9K52DIHXTDSYU · 0.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis.
Experimental design: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates.
Results: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts.
Conclusions and clinical relevance: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.
[Show abstract][Hide abstract] ABSTRACT: The ability to transfer image markup and annotation data from one scanned image of a slide to a newly acquired image of the same slide within a single vendor platform was investigated. The goal was to study the ability to use image markup and annotation data files as a mechanism to capture and retain pathologist knowledge without retaining the entire whole slide image (WSI) file.
Accepted mathematical principles were investigated as a method to overcome variations in scans of the same glass slide and to accurately associate image markup and annotation data across different WSI of the same glass slide. Trilateration was used to link fixed points within the image and slide to the placement of markups and annotations of the image in a metadata file.
Variation in markup and annotation placement between WSI of the same glass slide was reduced from over 80 μ to less than 4 μ in the x-axis and from 17 μ to 6 μ in the y-axis (P < 0.025).
This methodology allows for the creation of a highly reproducible image library of histopathology images and interpretations for educational and research use.
[Show abstract][Hide abstract] ABSTRACT: We describe the transfer of blaKPC-4 from Enterobacter cloacae to Serratia marcescens in a single patient. DNA sequencing revealed that KPC-4 was encoded on an IncL/M plasmid, pNE1280, closely related to pCTX-M360.
Further analysis found that KPC-4 was encoded within a novel Tn4401 element (Tn4401f) containing a truncated tnpA and lacking tnpR, ISKpn7 left, and Tn4401 IRL-1, which are conserved in other Tn4401 transposons. This study highlights the continued evolution of Tn4401 transposons and movement to multiple plasmid backbones that results in acquisition by multiple species of Gram-negative bacilli.
[Show abstract][Hide abstract] ABSTRACT: A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to demonstrate a prototype tool for measuring infectivity of an aerosolized human pathogen - influenza A/PR/8/34 (H1N1) virus - using a small-animal model in the Controlled Aerosol Test System (CATS).
Intranasal inoculation of nonadapted H1N1 virus into C57BL, BALB/c and CD-1 mice caused infection in all three species. Respiratory exposure of CD-1 mice to the aerosolized virus at graduated doses was accomplished in a modified rodent exposure apparatus. Weight change was recorded for 7 days postexposure, and viral populations in lung tissue homogenates were measured post mortem by DNA amplification (qRT-PCR), direct fluorescence and microscopic evaluation of cytopathic effect. Plots of weight change and of PCR cycle threshold vs delivered dose were linear to threshold doses of ~40 TCID(50) and ~12 TCID(50) , respectively.
MID(50) for inspired H1N1 aerosols in CD-1 mice is between 12 and 40 TCID(50) ; proportionality to dose of weight loss and viral populations makes the CD-1 mouse a useful model for measuring infectivity by inhalation.
In the CATS, this mouse-virus model provides the first quantitative method to evaluate the ability of respiratory protective technologies to attenuate the infectivity of an inspired pathogenic aerosol.
[Show abstract][Hide abstract] ABSTRACT: We investigated the use of whole-genome mapping and pulsed-field gel electrophoresis (PFGE) with isolates from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and whole-genome mapping were concordant with 22 of 23 isolates. Whole-genome mapping is a viable alternative tool for the epidemiological analysis of Salmonella food-borne disease investigations.
[Show abstract][Hide abstract] ABSTRACT: The use of high-resolution digital images of histopathology slides as a routine diagnostic tool for surgical pathology was investigated. The study purpose was to determine the diagnostic concordance between pathologic interpretations using whole-slide imaging and standard light microscopy. Two hundred fifty-one consecutive surgical pathology cases (312 parts, 1085 slides) from a single pathology service were included in the study after cases had been signed out and reports generated. A broad array of diagnostic challenges and tissue sources were represented, including 52 neoplastic cases. All cases were digitized at ×20 and presented to 2 pathologists for diagnosis using whole-slide imaging as the sole diagnostic tool. Diagnoses rendered by the whole-slide imaging pathologists were compared with the original light microscopy diagnoses. Overall concordance between whole-slide imaging and light microscopy as determined by a third pathologist and jury panel was 96.5% (95% confidence interval, 94.8%-98.3%). Concordance between whole-slide imaging pathologists was 97.7% (95% confidence interval, 94.7%-99.2%). Five cases were discordant between the whole-slide imaging diagnosis and the original light microscopy diagnosis, of which 2 were clinically significant. Discordance resulted from interpretive criteria or diagnostic error. The whole-slide imaging modality did not contribute to diagnostic differences. Problems encountered by the whole-slide imaging pathologists primarily involved the inability to clearly visualize nuclear detail or microscopic organisms. Technical difficulties associated with image scanning required at least 1 slide be rescanned in 13% of the cases. Technical and operational issues associated with whole-slide imaging scanning devices used in this study were found to be the most significant obstacle to the use of whole-slide imaging in general surgical pathology.
Human pathology 05/2012; 43(10):1739-44. DOI:10.1016/j.humpath.2011.12.023 · 2.77 Impact Factor