Abstract
Objectives
To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2.
Methods
The most efficient pool size was determined to be five specimens ...using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools.
Results
All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions.
Conclusions
When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.
Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 ...produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Computational analysis of metagenomes requires the taxonomical assignment of the genome contigs assembled from DNA reads of environmental samples. Because of the diverse nature of microbiomes, the ...length of the assemblies obtained can vary between a few hundred bp to a few hundred Kbp. Current taxonomic classification algorithms provide accurate classification for long contigs or for short fragments from organisms that have close relatives with annotated genomes. These are significant limitations for metagenome analysis because of the complexity of microbiomes and the paucity of existing annotated genomes.
We propose a robust taxonomic classification method, RAIphy, that uses a novel sequence similarity metric with iterative refinement of taxonomic models and functions effectively without these limitations. We have tested RAIphy with synthetic metagenomics data ranging between 100 bp to 50 Kbp. Within a sequence read range of 100 bp-1000 bp, the sensitivity of RAIphy ranges between 38%-81% outperforming the currently popular composition-based methods for reads in this range. Comparison with computationally more intensive sequence similarity methods shows that RAIphy performs competitively while being significantly faster. The sensitivity-specificity characteristics for relatively longer contigs were compared with the PhyloPythia and TACOA algorithms. RAIphy performs better than these algorithms at varying clade-levels. For an acid mine drainage (AMD) metagenome, RAIphy was able to taxonomically bin the sequence read set more accurately than the currently available methods, Phymm and MEGAN, and more accurately in two out of three tests than the much more computationally intensive method, PhymmBL.
With the introduction of the relative abundance index metric and an iterative classification method, we propose a taxonomic classification algorithm that performs competitively for a large range of DNA contig lengths assembled from metagenome data. Because of its speed, simplicity, and accuracy RAIphy can be successfully used in the binning process for a broad range of metagenomic data obtained from environmental samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human BChE (butyrylcholinesterase) 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, rBChE (recombinant BChE) 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 15-50 consecutive proline residues.
Francisella tularensis
is a highly infectious zoonotic pathogen with as few as 10 organisms causing tularemia, a disease that is fatal if untreated. Although
F. tularensis
subspecies
tularensis
(type ...A) and subspecies
holarctica
(type B) share over 99.5% average nucleotide identity, notable differences exist in genomic organization and pathogenicity. The type A clade has been further divided into subtypes A.I and A.II, with A.I strains being recognized as some of the most virulent bacterial pathogens known. In this study, we report on major disparities that exist between the
F. tularensis
subpopulations in arginine catabolism and subsequent polyamine biosynthesis. The genes involved in these pathways include the
speHEA
and
aguAB
operons, along with
metK
. In the hypervirulent
F. tularensis
A.I clade, such as the A.I prototype strain SCHU S4, these genes were found to be intact and highly transcribed. In contrast, both subtype A.II and type B strains have a truncated
speA
gene, while the type B clade also has a disrupted
aguA
and truncated
aguB
. Ablation of the chromosomal
speE
gene that encodes a spermidine synthase reduced subtype A.I SCHU S4 growth rate, whereas the growth rate of type B LVS was enhanced. These results demonstrate that spermine synthase SpeE promotes faster replication in the
F. tularensis
A.I clade, whereas type B strains do not rely on this enzyme for
in vitro
fitness. Our ongoing studies on amino acid and polyamine flux within hypervirulent A.I strains should provide a better understanding of the factors that contribute to
F. tularensis
pathogenicity.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The 2014–2016 West Africa Ebola virus (EBOV) outbreak coupled with the most recent outbreaks in Central Africa underscore the need to develop effective treatment strategies against EBOV. Although ...several therapeutic options have shown great potential, developing a wider breadth of countermeasures would increase our efforts to combat the highly lethal EBOV. Here we show that human cathelicidin antimicrobial peptide (AMP) LL-37 and engineered LL-37 AMPs inhibit the infection of recombinant virus pseudotyped with EBOV glycoprotein (GP) and the wild-type EBOV. These AMPs target EBOV infection at the endosomal cell-entry step by impairing cathepsin B-mediated processing of EBOV GP. Furthermore, two engineered AMPs containing D-amino acids are particularly potent in blocking EBOV infection in comparison with other AMPs, most likely owing to their resistance to intracellular enzymatic degradation. Our results identify AMPs as a novel class of anti-EBOV therapeutics and demonstrate the feasibility of engineering AMPs for improved therapeutic efficacy.
Display omitted
•Cathelicidin-derived antimicrobial peptides (AMPs) potently inhibit EBOV infection•D-form AMPs are more resistant to proteolytic cleavage than L-form AMPs in the cell•AMPs prevent cathepsin B-mediated processing of EBOV GP1, 2
Drugs; Molecular Biology; Viral Microbiology
Summary 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.
Objectives:
Public health laboratories (PHLs) are essential components of US Public Health Service operations. The health information technology that supports PHLs is central to effective and ...efficient laboratory operations and overall public health response to infectious disease management. This analysis presents key information on how the Nebraska Public Health Laboratory (NPHL) information technology system evolved to meet the demands of the COVID-19 pandemic.
Materials and Methods:
COVID-19 presented numerous, unforeseen information technology system challenges. The most notable challenges requiring changes to NPHL software systems and capability were improving efficiency of the laboratory operation due to high-volume testing, responding daily to demands for timely data for analysis by partner systems, interfacing with multiple testing (equipment) platforms, and supporting community-based specimen collection programs.
Results:
Improvements to the NPHL information technology system enabled NPHL to perform >121 000 SARS-CoV-2 polymerase chain reaction tests from March 2020 through January 2022 at a sustainable rate of 2000 SARS-CoV-2 tests per day, with no increase in laboratory staffing. Electronic reporting of 62 000 rapid antigen tests eliminated paper reporting and extended testing services throughout the state. Collection of COVID-19 symptom data before specimen collection enabled NPHL to make data-driven decisions to perform pool testing and conserve testing kits when supplies were low.
Practice Implications:
NPHL information technology applications proved essential for managing health care provider workload, prioritizing the use of scarce testing supplies, and managing Nebraska’s overall pandemic response. The NPHL experience provides useful examples of a highly capable information technology system and suggests areas for additional attention in the PHL environment, including a focus on end users, collaboration with various partners, and investment in information technology.
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