RESUMO
Acute gastroenteritis (AGE) is a leading cause of morbidity and mortality worldwide across all age groups that disproportionally affects young children in low- and middle-income countries and immunocompromised patients in high-income countries. Regional outbreaks of AGE are typically detected by traditional microbiological detection methods that target limited organisms and are associated with low sensitivity and lengthy time-to-results. Combined, these may result in repeat testing, imprecise or delayed treatment, and delayed recognition of outbreaks. We conducted a multi-site prospective study comparing the BioCode Gastrointestinal Pathogen Panel (BioCode GPP) for the detection of 17 common bacterial, viral, and protozoan causes of gastroenteritis with reference methods, including stool culture, enzyme immunoassays, pathogen-specific PCR assays, and sequencing. One thousand five hundred fifty-eight residual, de-identified stool samples (unpreserved stool and stool in Cary-Blair transport medium) were enrolled and tested for 11 bacterial, 3 viral, and 3 protozoan pathogens. BioCode GPP and reference methods were positive for 392 (25.2%) and 283 (18.2%) samples, respectively (P < 0.0001). In this study, the BioCode GPP and reference methods detected 69 and 65 specimens positive for Clostridioides difficile, 51 and 48 for enteroaggregative Escherichia coli, 33 and 27 for enterotoxigenic E. coli, 50 and 47 for norovirus GI/GII, and 30 and 22 for rotavirus A, respectively. The BioCode GPP showed good positive and negative agreements for each pathogen ranging from 89.5% to 100%, with overall sensitivity and specificity of 96.1% and 99.7%, post adjudication. The BioCode GPP detected >1 pathogens in 49 samples, representing 12.5% of the total 392 positive specimens. IMPORTANCE: This study highlights performance of a novel technology for timely and accurate detection and differentiation of 17 common bacterial, viral, and protozoan causes of gastroenteritis. Utilizing molecular tests such as the BioCode Gastrointestinal Pathogen Panel may improve the detection of gastrointestinal pathogens and provide actionable results, particularly for patient populations at most risk.
Assuntos
Bacteriófagos , Escherichia coli Enterotoxigênica , Gastroenterite , Norovirus , Rotavirus , Humanos , Diarreia/diagnóstico , Fezes/microbiologia , Gastroenterite/diagnóstico , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) utilizes reverse transcriptase PCR (RT-PCR) in combination with barcoded magnetic beads to amplify, detect, and identify respiratory pathogens. This panel qualitatively detects and identifies 14 viruses, including influenza virus A with H1 pdm09, H1, and H3 subtyping; influenza B; respiratory syncytial virus (RSV); human metapneumovirus; parainfluenza virus 1; parainfluenza virus 2; parainfluenza virus 3; parainfluenza virus 4; coronavirus (229E, NL63, OC43, and HKU1); adenovirus; and human rhinovirus/enterovirus, and 3 bacteria, including Chlamydia pneumoniae, Mycoplasma pneumoniae, and Bordetella pertussis. Reproducibility, which was assessed with contrived specimens containing 12 targets at 3 clinical sites, with 2 operators at each site for 5 days, was 99.4% for Flu A H3 and Flu B, 98.9% for RSV, and 100% for the remaining 9 targets assayed. A multicenter clinical trial evaluated the performance of the BioCode RPP with 2,647 nasopharyngeal swab specimens from 5 geographically distinct sites and revealed comparable performance between the BioCode RPP and FilmArray Respiratory Panel (FA-RP). Specifically, the positive percent agreements (PPAs) for various pathogens ranged between 80.8% and 100% compared with the FA-RP (1.7 and 2.0). Negative percent agreement ranged from 98.4% to 100% for BioCode RPP. The BioCode RPP also offers scalable automated testing capability of up to 96 specimens in a single run with total sample-to-result time under 5 h. The invalid rate of the BioCode RPP on initial testing was 1.0% (26/2,649). IMPORTANCE Early detection of microbial pathogens causing respiratory tract infection plays a crucial role in clinical management. The BioCode Respiratory Pathogen Panel (BioCode RPP) is a high-throughput test that utilizes RT-PCR in combination with barcoded magnetic beads to amplify, detect, and identify 17 respiratory pathogens, including 14 viruses and 3 bacteria. This study summarizes data generated from a multicenter clinical trial evaluating the performance of the BioCode RPP on 2,647 nasopharyngeal swab specimens from five geographically distinct sites.
Assuntos
Infecções por Paramyxoviridae , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Viroses , Vírus , Humanos , Viroses/diagnóstico , Reprodutibilidade dos Testes , Vírus/genética , Bactérias , Infecções Respiratórias/microbiologia , NasofaringeRESUMO
Synthetic elicitors are drug-like compounds that induce plant immune responses but are structurally distinct from natural defense elicitors. Using high-throughput screening, we previously identified 114 synthetic elicitors that activate the expression of a pathogen-responsive reporter gene in Arabidopsis (Arabidopsis thaliana). Here, we report on the characterization of one of these compounds, 2-(5-bromo-2-hydroxy-phenyl)-thiazolidine-4-carboxylic acid (BHTC). BHTC induces disease resistance of plants against bacterial, oomycete, and fungal pathogens and has a unique mode of action and structure. Surprisingly, we found that low doses of BHTC enhanced root growth in Arabidopsis, while high doses of this compound inhibited root growth, besides inducing defense. These effects are reminiscent of the hormetic response, which is characterized by low-dose stimulatory effects of a wide range of agents that are toxic or inhibitory at higher doses. Like its effects on defense, BHTC-induced hormesis in Arabidopsis roots is partially dependent on the WRKY70 transcription factor. Interestingly, BHTC-induced root hormesis is also affected in the auxin-response mutants axr1-3 and slr-1. By messenger RNA sequencing, we uncovered a dramatic difference between transcriptional profiles triggered by low and high doses of BHTC. Only high levels of BHTC induce typical defense-related transcriptional changes. Instead, low BHTC levels trigger a coordinated intercompartmental transcriptional response manifested in the suppression of photosynthesis- and respiration-related genes in the nucleus, chloroplasts, and mitochondria as well as the induction of development-related nuclear genes. Taken together, our functional characterization of BHTC links defense regulation to hormesis and provides a hypothetical transcriptional scenario for the induction of hormetic root growth.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/imunologia , Hormese , Tiazolidinas/farmacologia , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Clorobenzoatos/química , Clorobenzoatos/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Peronospora/patogenicidade , Doenças das Plantas/microbiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Relação Estrutura-Atividade , Tiazolidinas/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologiaRESUMO
Transgenic Arabidopsis seedlings containing a pathogen-responsive reporter gene allow for convenient high-throughput screening of chemical libraries for compounds that induce plant defense responses. Candidates identified by such screens can be further tested for their ability to protect plants from pathogen-caused diseases. Using Arabidopsis defense signaling mutants, defined regulatory processes that are targeted by a given candidate molecule can be easily narrowed down. Here, we provide a detailed high-throughput screening protocol for library compounds that activate a pathogen-responsive reporter gene in liquid-grown Arabidopsis seedlings.
Assuntos
Arabidopsis/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Plântula/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter , Ensaios de Triagem em Larga Escala , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/imunologia , Plântula/genética , Plântula/imunologia , Bibliotecas de Moléculas PequenasRESUMO
Immune responses of Arabidopsis (Arabidopsis thaliana) are at least partially mediated by coordinated transcriptional up-regulation of plant defense genes, such as the Late/sustained Up-regulation in Response to Hyaloperonospora parasitica (LURP) cluster. We found a defined region in the promoter of the LURP member CaBP22 to be important for this response. Using a CaBP22 promoter-reporter fusion, we have established a robust and specific high-throughput screening system for synthetic defense elicitors that can be used to trigger defined subsets of plant immune responses. Screening a collection of 42,000 diversity-oriented molecules, we identified 114 candidate LURP inducers. One representative, 3,5-dichloroanthranilic acid (DCA), efficiently induced defense reactions to the phytopathogens H. parasitica and Pseudomonas syringae. In contrast to known salicylic acid analogs, such as 2,6-dichloroisonicotinic acid (INA), which exhibit a long-lasting defense-inducing activity and are fully dependent on the transcriptional cofactor NPR1 (for Nonexpresser of Pathogenesis-Related genes1), DCA acts transiently and is only partially dependent on NPR1. Microarray analyses revealed a cluster of 142 DCA- and INA-responsive genes that show a pattern of differential expression coinciding with the kinetics of DCA-mediated disease resistance. These ACID genes (for Associated with Chemically Induced Defense) constitute a core gene set associated with chemically induced disease resistance, many of which appear to encode components of the natural immune system of Arabidopsis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Calmodulina/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , ortoaminobenzoatos/farmacologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Calmodulina/química , Calmodulina/genética , Clorobenzoatos , Perfilação da Expressão Gênica , Genes Reporter , Imunidade Inata/genética , Cinética , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Oomicetos/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Pseudomonas syringae/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/análise , Ácido Salicílico/farmacologia , ortoaminobenzoatos/químicaRESUMO
LURP1 is a member of the LURP cluster and the PR1 regulon, two previously defined sets of co-expressed Arabidopsis thaliana genes that share a pronounced upregulation in response to infections by the pathogenic oomycete Hyaloperonospora parasitica. LURP1 shows the most extreme transcriptional inducibility by H. parasitica of all LURP and PR1 regulon genes. Using insertion mutants we found that LURP1 is required for full basal defense to H. parasitica and resistance to this pathogen mediated by the R-proteins RPP4 and RPP5. The LURP1 protein shows no obvious similarity to proteins of known molecular function. We identified a 39-bp region of the LURP1 promoter that mediates reporter gene expression in response to H. parasitica and salicylic acid. This promoter region contains a W box motif, W(LURP1), that interacts in vitro with nuclear factors producing two separate DNA-binding patterns. W(LURP1)-related sequences are strongly enriched in the promoters of the PR1 regulon, suggesting a role for this motif in the coordinated expression of genes inducible by H. parasitica and related defense conditions.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/imunologia , Interações Hospedeiro-Patógeno , Oomicetos/imunologia , Doenças das Plantas/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutagênese Insercional , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Fatores de Transcrição/metabolismoRESUMO
AtWRKY70, encoding a WRKY transcription factor, is co-expressed with a set of Arabidopsis genes that share a pattern of RPP4- and RPP7-dependent late upregulation in response to Hyaloperonospora parasitica infection (LURP) genes. We show that AtWRKY70 is required for both full RPP4-mediated resistance and basal defense against H. parasitica. These two defense pathways are related to each other, because they require PAD4 and salicylic acid (SA). RPP7 function, which is independent from PAD4 and SA, is not affected by insertions in AtWRKY70. Although AtWRKY70 is required for RPP4-resistance, it appears not to contribute significantly to RPP4-triggered cell death. Furthermore, our data indicate that AtWRKY70 functions downstream of defense-associated reactive oxygen intermediates and SA. Constitutive and RPP4-induced transcript levels of two other LURP genes are reduced in AtWRKY70 T-DNA mutants, indicating a direct or indirect role for AtWRKY70 in their regulation. We propose that AtWRKY70 is a component of a basal defense mechanism that is boosted by engagement of either RPP4 or RPP7 and is required for RPP4-mediated resistance.