Detection of botulinum neurotoxin or isolation of the toxin-producing organism is required for the laboratory confirmation of botulism in clinical specimens. In an effort to reduce animal testing ...required by the gold standard method of botulinum neurotoxin detection, the mouse bioassay, many technologies have been developed to detect and characterize the causative agent of botulism. Recent advancements in these technologies have led to improvements in technical performance of diagnostic assays; however, many emerging assays have not been validated for the detection of all serotypes in complex clinical and environmental matrices. Improvements to culture protocols, endopeptidase-based assays, and a variety of immunological and molecular methods have provided laboratories with a variety of testing options to evaluate and incorporate into their testing algorithms. While significant advances have been made to improve these assays, additional work is necessary to evaluate these methods in various clinical matrices and to establish standardized criteria for data analysis and interpretation.
Coxiella burnetii, the causative agent of Q fever, is a long-standing public health problem. Infected animals shed the organism, resulting in aerosol transmission to humans. This organism can ...potentially be used as a bioterrorism weapon and is on the Department of Health and Human Service Select Agent List. Assay development for detecting C. burnetii in environmental samples has been limited.
We describe the use of Standard Method Performance Requirements (SMPR®) 2015.011 to detect Coxiella in air filters and liquids to validate additional environmental samples.
SMPR 2015.011 was used to validate a real-time polymerase chain reaction (rtPCR) assay developed to detect C. burnetii DNA in powder samples submitted to the public health laboratory for biothreat analysis.
Our laboratory developed an assay to detect the icd gene of C. burnetii. The LOD for the assay was 33 gene copies per rtPCR reaction in buffer and 260 in each of the three separate powdered samples.
The SMPR 2015.011 allowed validation of an assay to detect Coxiella nucleic acid in an environmental sample. The assay was sensitive, robust, specific, and able to detect this select agent in powders.
Development of detection assays for agents that are difficult to culture and have limited validation material available can be problematic for manufacturers. Using the SMPR 2015.011 developed for the detection of Coxiella as well as the SMPR 2016.012 for the detection of Variola, we demonstrated that assays can be appropriately validated using alternative approaches.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may ...trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected
mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection).
The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.
Currently, the gold standard method for active botulinum neurotoxin (BoNT) detection is the mouse bioassay (MBA). A Centers for Disease Control and Prevention-developed mass spectrometry (MS)-based ...assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD) and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD
, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD
, somewhat more sensitive than the MS method of 18 mLD
. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.
To minimize specimen volume, handling and testing time, we have developed two TaqMan® multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin ...production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8–20 cfu per rtPCR for the clinical stool matrix.
•We developed two PCR assays to detect Staphylococcal enterotoxins A-E in specimens.•The multiplex PCR assays also detect Toxic Shock Syndrome Toxin production genes.•The assays were validated using stool specimens and clinical isolates.•This automated method uses minimal sample volume and reduces reagent costs.
produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when
colonizes the ...intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by
as their intestinal microbiota is not fully developed and provides little competition, allowing
to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (
) gene. One of the most recently recognized subtypes for
/B is subtype
/B7. We identified through whole genome sequencing five
isolates harboring
/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these
subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.
During February and March 2010, the New York State Department of Health investigated secondary and tertiary vaccinia contact transmission from a military vaccinee to 4 close contacts. Identification ...of these cases underscores the need for strict adherence to postvaccination infection control guidance to avoid transmission of the live virus.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has ...been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.
Presents the results of a qualitative interpretive case study of three New Zealand early childhood education (ECE) services to determine how each service practices educational leadership. Looks at ...how these services use internal evaluation processes; explores factors challenging or enabling teacher involvement and practice of educational leadership through internal evaluation processes; and to ascertain how services monitor the effect of changes on teaching practice following an internal evaluation. Identifies seven effective leadership practices. Source: National Library of New Zealand Te Puna Matauranga o Aotearoa, licensed by the Department of Internal Affairs for re-use under the Creative Commons Attribution 3.0 New Zealand Licence.
Abstract
Background
The US’ coronavirus disease 2019 (COVID-19) epidemic has grown extensively since February 2020, with substantial associated hospitalizations and mortality; New York State has ...emerged as the national epicenter. We report on the extent of testing and test results during the month of March in New York State, along with risk factors, outcomes, and household prevalence among initial cases subject to in-depth investigations.
Methods
Specimen collection for COVID-19 testing was conducted in healthcare settings, community-based collection sites, and by home testing teams. Information on demographics, risk factors, and hospital outcomes of cases was obtained through epidemiological investigations and an electronic medical records match, and summarized descriptively. Active testing of initial case’s households enabled estimation of household prevalence.
Results
During March in New York State, outside of New York City, a total of 47 326 persons tested positive for severe acute respiratory syndrome coronavirus 2, out of 141 495 tests (33% test-positive), with the highest number of cases located in the metropolitan region counties. Among 229 initial cases diagnosed through 12 March, by 30 March 13% were hospitalized and 2% died. Testing conducted among 498 members of these case’s households found prevalent infection among 57%, excluding first-reported cases 38%. In these homes, we found a significant age gradient in prevalence, from 23% among those < 5 years to 68% among those ≥ 65 years (P < .0001).
Conclusions
New York State faced a substantial and increasing COVID-19 outbreak during March 2020. The earliest cases had high levels of infection in their households and by the end of the month, the risks of hospitalization and death were high.
We detail the extent of testing and positive results for coronavirus disease 2019 during the month of March, 2020 in New York State, a focal point of the US epidemic, and provide details on initial cases, including household prevalence.
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