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1.
J Clin Virol ; 124: 104262, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32007841

RESUMO

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories.


Assuntos
Surtos de Doenças , Enterovirus Humano D , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/epidemiologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Algoritmos , Criança , Infecções por Enterovirus/virologia , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Humanos , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Estados Unidos/epidemiologia
2.
JMIR Public Health Surveill ; 4(3): e59, 2018 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-29980501

RESUMO

BACKGROUND: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

3.
J Pediatric Infect Dis Soc ; 6(3): 231-238, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27147712

RESUMO

BACKGROUND: Diarrheal diseases are a major cause of ambulatory care visits and hospitalizations among children. Because of overlapping signs and symptoms and expensive and inefficient testing methods, the etiology of pediatric diarrhea is rarely established. METHODS: We identified children <18 years of age who were evaluated for diarrhea at Primary Children's Hospital in Salt Lake City, Utah, between October 2010 and September 2012. Stool specimens submitted for testing were evaluated by using the FilmArray gastrointestinal diagnostic system, which is a rapid multiplex polymerase chain reaction platform that can simultaneously detect 23 bacterial, viral, and protozoal agents. RESULTS: A pathogen was detected in 561 (52%) of 1089 diarrheal episodes. The most commonly detected pathogens included toxigenic Clostridium difficile (16%), diarrheagenic Escherichia coli (15%), norovirus GI/GII (11%), and adenovirus F 40/41 (7%). Shiga toxin-producing E coli were detected in 43 (4%) specimens. Multiple pathogens were identified in 160 (15%) specimens. Viral pathogens (norovirus, adenovirus, rotavirus, and sapovirus) were more common among children <5 years old than among those 5 to 17 years old (38% vs 16%, respectively; P < .001). Bacterial pathogens were identified most commonly in children 2 to 4 years of age. Children with 1 or more underlying chronic medical conditions were less likely to have a pathogen identified than those without a chronic medical condition (45% vs 60%, respectively; P < .01). Viral pathogens were detected more commonly in the winter, whereas bacterial pathogens were detected more commonly in the summer. CONCLUSIONS: Toxigenic C difficile, diarrheagenic E coli, and norovirus were the leading organisms detected among these children with diarrhea. Viral pathogens are identified frequently among young children with acute gastroenteritis.


Assuntos
Diarreia/microbiologia , Adolescente , Criança , Pré-Escolar , Diarreia/parasitologia , Diarreia/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase Multiplex , Estações do Ano , Utah/epidemiologia
4.
J Mol Diagn ; 16(2): 159-62, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24457120

RESUMO

This Commentary highlights the article by Satterfield that describes a new class of primer technology-cooperative primers, which prevent primer-dimer amplification.


Assuntos
Primers do DNA/normas , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Humanos
5.
Diagn Microbiol Infect Dis ; 74(4): 349-55, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22999332

RESUMO

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.


Assuntos
Bacteriemia/diagnóstico , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Adulto , Bacteriemia/microbiologia , Bactérias/genética , Criança , Pré-Escolar , Farmacorresistência Bacteriana , Humanos
6.
J Clin Microbiol ; 50(10): 3216-21, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22814461

RESUMO

Respiratory virus infections cause significant morbidity and mortality in immunocompromised patients. Timely diagnosis is needed to provide optimal clinical care. Diagnostic tests routinely available at most institutions are limited by poor sensitivity and a slow turnaround time. We collected 90 respiratory samples from 87 immunocompromised patients (56 bronchoalveolar lavage and 34 nasopharyngeal aspirate samples) in order to compare the performance of routine respiratory virus testing available at our institution to the FilmArray respiratory panel assay, a novel diagnostic tool which utilizes multiplex PCR to test for 21 respiratory pathogens with a 1-h turnaround time. Samples with discordant results and 13 samples with concordant results underwent further verification testing by laboratory-developed real-time PCR. The FilmArray assay identified viral pathogens in more samples than did clinical testing (30/90 versus 16/90; McNemar P = 0.001). Most of the additional viral pathogens identified by the FilmArray respiratory panel assay that were confirmed by verification testing were pathogens not assessed by routine clinical tests, including rhinovirus/enterovirus, human metapneumovirus, and coronavirus. The FilmArray respiratory panel assay allowed for increased identification of respiratory viral pathogens in this cohort of immunocompromised patients.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/diagnóstico , Viroses/diagnóstico , Vírus/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Virologia/métodos , Vírus/classificação , Vírus/genética , Adulto Jovem
7.
PLoS One ; 6(10): e26047, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22039434

RESUMO

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.


Assuntos
Infecções Respiratórias/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos
8.
J Clin Virol ; 52(3): 210-4, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21855405

RESUMO

BACKGROUND: Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods. OBJECTIVE: Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR. STUDY DESIGN: Children aged 1 month-17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard. RESULTS: 285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods. CONCLUSIONS: Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Líquido da Lavagem Nasal/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Sistema Respiratório/virologia , Infecções Respiratórias/diagnóstico , Rhinovirus/isolamento & purificação , Viroses/diagnóstico , Adolescente , Criança , Pré-Escolar , Face/virologia , Feminino , Humanos , Lactente , Masculino , Nasofaringe/virologia , Infecções por Picornaviridae/diagnóstico , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sinciciais Respiratórios/genética , Infecções Respiratórias/virologia , Rhinovirus/genética , Sensibilidade e Especificidade , Manejo de Espécimes , Viroses/virologia
9.
Pediatr Infect Dis J ; 30(4): 289-94, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21057372

RESUMO

BACKGROUND: Parapneumonic empyema (PPE) is an increasingly common complication of bacterial pneumonia. Epidemiologic study is complicated by the low frequency of positive cultures. We sought to describe the epidemiology of PPE in children using molecular analysis of pleural fluid. METHODS: We performed molecular testing for bacterial pathogens using archived pleural fluid from children hospitalized in 2009 with PPE. Real-time polymerase chain reaction (PCR) to detect Streptococcus pneumoniae, Staphylococcus aureus (including methicillin-resistant), Streptococcus pyogenes, Haemophilus influenzae, and Mycoplasma pneumoniae as well as PCR-based serotyping of S. pneumoniae was performed. Demographic, laboratory, and microbiologic data were abstracted. RESULTS: Pleural fluid specimens from 63 children were available for PCR. By culture, a pathogen was isolated from blood and/or pleural fluid in 22 (35%) patients, with S. pneumoniae in 15 (24%), S. pyogenes in 3 (5%), and methicillin-resistant Staphylococcus aureus in 4 (6%). By PCR, a pathogen was detected in 53 (84%), including S. pneumoniae in 45 (71%). Compared with culture, PCR testing significantly increased detection of any pathogen (35% vs. 84%; P < 0.001) and of S. pneumoniae (24% vs. 71%; P < 0.001). Serotype 7F was the most common pneumococcal serotype detected. Comparison of culture-negative to culture-positive disease showed differences in both the pathogen profile and clinical outcomes. CONCLUSIONS: Molecular analysis of pleural fluid more than doubled the detection of pathogens causing PPE. S. pneumoniae was the most common cause of both culture-positive and culture-negative PPE, although serotype distribution and outcomes differed.


Assuntos
Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Técnicas Bacteriológicas/métodos , Empiema Pleural/epidemiologia , Empiema Pleural/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Masculino , Mycoplasma pneumoniae/isolamento & purificação , Prevalência , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pyogenes/isolamento & purificação
10.
Clin Chem ; 54(10): 1648-56, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18676584

RESUMO

BACKGROUND: DNA hairpins have been used in molecular analysis of PCR products as self-probing amplicons. Either physical separation or fluorescent oligonucleotides with covalent modifications were previously necessary. METHODS: We performed asymmetric PCR for 40-45 cycles in the presence of the saturating DNA dye, LCGreen Plus, with 1 primer including a 5' tail complementary to its extension product, but without any special covalent modifications. Samples were amplified either on a carousel LightCycler for speed or on a 96/384 block cycler for throughput. In addition to full-length amplicon duplexes, single-stranded hairpins were formed by the primer tail "snapping back" and hybridizing to its extension product. High-resolution melting was performed on a HR-1 (for capillaries) or a LightScanner (for plates). RESULTS: PCR products amplified with a snapback primer showed both hairpin melting at lower temperature and full-length amplicon melting at higher temperature. The hairpin melting temperature was linearly related to the stem length (6-28 bp) and inversely related to the log of the loop size (17-135 bases). We easily genotyped heterozygous and homozygous variants within the stem, and 100 blinded clinical samples previously typed for F5 1691G>A (Leiden) were completely concordant by snapback genotyping. We distinguished 7 genotypes in 2 regions of CFTR exon 10 with symmetric PCR using 2 snapback primers followed by product dilution to favor intramolecular hybridization. CONCLUSIONS: Snapback primer genotyping with saturating dyes provides the specificity of a probe with only 2 primers that are free of special covalent labels in a closed-tube system.


Assuntos
Corantes/química , DNA/genética , Sequência de Bases , DNA/química , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples
11.
Virology ; 313(1): 170-83, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12951031

RESUMO

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.


Assuntos
Antivirais/genética , Peptídeos/genética , Rhinovirus/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/isolamento & purificação , Antivirais/farmacologia , Sequência de Bases , Clonagem Molecular/métodos , Efeito Citopatogênico Viral/efeitos dos fármacos , DNA Complementar/biossíntese , DNA Complementar/genética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Biblioteca Gênica , Vetores Genéticos , Células HeLa , Humanos , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Placenta/química , Retroviridae/genética , Rhinovirus/fisiologia , Transfecção , Replicação Viral/efeitos dos fármacos
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