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1.
Microb Pathog ; 126: 292-297, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30414838

RESUMO

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.


Assuntos
Bactérias/química , Técnicas Bacteriológicas/métodos , DNA Bacteriano/isolamento & purificação , Bactérias/genética , Parede Celular/química , DNA Bacteriano/química , DNA Bacteriano/genética , Detergentes , Biologia Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Saliva/microbiologia , Esporos Bacterianos/química , Esporos Bacterianos/genética , Escarro/microbiologia
2.
Appl Environ Microbiol ; 77(24): 8625-34, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22003031

RESUMO

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.


Assuntos
Clostridium botulinum tipo E/classificação , Clostridium botulinum tipo E/genética , DNA Bacteriano/genética , Tipagem Molecular/métodos , Polimorfismo Genético , Toxinas Botulínicas/genética , Botulismo/microbiologia , Clostridium botulinum tipo E/isolamento & purificação , Análise por Conglomerados , Elementos de DNA Transponíveis , Microbiologia Ambiental , Microbiologia de Alimentos , Genótipo , Humanos , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNA
3.
Infect Immun ; 77(11): 4859-67, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19703971

RESUMO

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos de Linfócito B/imunologia , Sequência de Aminoácidos , Animais , Antraz/prevenção & controle , Vacinas contra Antraz/imunologia , Afinidade de Anticorpos , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
4.
BMJ Glob Health ; 4(Suppl 2): e001179, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30815287

RESUMO

Diagnostics are fundamental for successful outbreak containment. In this supplement, 'Diagnostic preparedness for WHO Blueprint pathogens', we describe specific diagnostic challenges presented by selected priority pathogens most likely to cause future epidemics. Some challenges to diagnostic preparedness are common to all outbreak situations, as highlighted by recent outbreaks of Ebola, Zika and yellow fever. In this article, we review these overarching challenges and explore potential solutions. Challenges include fragmented and unreliable funding pathways, limited access to specimens and reagents, inadequate diagnostic testing capacity at both national and community levels of healthcare and lack of incentives for companies to develop and manufacture diagnostics for priority pathogens during non-outbreak periods. Addressing these challenges in an efficient and effective way will require multiple stakeholders-public and private-coordinated in implementing a holistic approach to diagnostics preparedness. All require strengthening of healthcare system diagnostic capacity (including surveillance and education of healthcare workers), establishment of sustainable financing and market strategies and integration of diagnostics with existing mechanisms. Identifying overlaps in diagnostic development needs across different priority pathogens would allow more timely and cost-effective use of resources than a pathogen by pathogen approach; target product profiles for diagnostics should be refined accordingly. We recommend the establishment of a global forum to bring together representatives from all key stakeholders required for the response to develop a coordinated implementation plan. In addition, we should explore if and how existing mechanisms to address challenges to the vaccines sector, such as Coalition for Epidemic Preparedness Innovations and Gavi, could be expanded to cover diagnostics.

5.
J Epidemiol Glob Health ; 6(4): 257-265, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27131103

RESUMO

This preliminary study evaluated the transport reagent OMNIgene SPUTUM (OMS) in a real-world, resource-limited setting: a zonal hospital and national tuberculosis (TB) reference laboratory, Nepal. The objectives were to: (1) assess the performance of OMS for transporting sputum from peripheral sites without cold chain stabilization; and (2) compare with Nepal's standard of care (SOC) for Mycobacterium tuberculosis smear and culture diagnostics. Sixty sputa were manually split into a SOC sample (airline-couriered to the laboratory, conventional processing) and an OMS sample (OMS added at collection, no cold chain transport or processing). Smear microscopy and solid culture were performed. Transport was 0-8days. Forty-one samples (68%) were smear-positive using both methods. Of the OMS cultures, 37 (62%) were positive, 22 (36%) were negative, and one (2%) was contaminated. Corresponding SOC results were 32 (53%), 21 (35%), and seven (12%). OMS "rescued" six (i.e., missed using SOC) compared with one rescue using SOC. Of smear-positives, six SOC samples produced contaminated cultures whereas only one OMS sample was contaminated. OMS reduced culture contamination from 12% to 2%, and improved TB detection by 9%. The results suggest that OMS could perform well as a no cold chain, long-term transport solution for smear and culture testing. The findings provide a basis for larger feasibility studies.


Assuntos
Refrigeração/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Humanos , Nepal
6.
Diagn Microbiol Infect Dis ; 86(3): 273-276, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27601382

RESUMO

OMNIgene®â€¢SPUTUM (OM-S) is a sputum transport reagent designed to work with all tuberculosis diagnostics and eliminate the need for cold chain. The aim of this preliminary study was to assess the compatibility of OM-S-treated sputum with the Xpert® MTB/RIF assay. Fifty-five characterized sputa from the FIND TB Specimen Bank were used. Compatibility of OM-S was assessed for both Xpert sample preparation methods: H.1 protocol (sediment, n=25) and H.2 protocol (direct expectorate, n=30). All controls were prepared using the H.2 protocol. Results revealed 100% concordance of MTB/RIF results for all except the low-positive group in the H.1 study arm (n=10; 88% concordance). OM-S-treated sputa were successful in both protocols; if the Xpert buffer is not added during the H.2 procedure, sample viscosity may require repeat testing. Using OM-S could offer users flexibility in clinical testing algorithms. Larger compatibility studies are warranted, particularly with respect to MTB/RIF results for low-positive samples.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Manejo de Espécimes/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Humanos , Indicadores e Reagentes
8.
Vector Borne Zoonotic Dis ; 14(4): 240-4, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24689928

RESUMO

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina G/sangue , Rickettsia prowazekii/imunologia , Sciuridae/microbiologia , Tifo Epidêmico Transmitido por Piolhos/epidemiologia , Animais , Reservatórios de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Rickettsia prowazekii/isolamento & purificação , Estudos Soroepidemiológicos , Tifo Epidêmico Transmitido por Piolhos/microbiologia , Adulto Jovem , Zoonoses
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