RESUMEN
The performance of Prove-it™ Bone&Joint, a novel microarray-based assay for direct pathogen detection from clinical samples was assessed. In culture-positive samples, Prove-it™ performed similarly with broad-range bacterial PCR in osteoarticular (sensitivity 62.9% vs. 57.7%) and non-osteoarticular samples (54.6% vs. 56.8%). Prove-it™ is a rapid tool providing preliminary results while awaiting culture results.
Asunto(s)
Artritis Infecciosa/diagnóstico , Artritis Infecciosa/microbiología , Bacterias/genética , Osteítis/diagnóstico , Osteítis/microbiología , Reacción en Cadena de la Polimerasa/métodos , Bacterias/clasificación , Bacterias/aislamiento & purificación , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The rapid identification of microbes responsible for bloodstream infections (BSIs) allows more focused and effective therapies and outcomes. DNA sequence-based methods offer an opportunity for faster, accurate diagnosis and for effective therapy. As our objective of the study, the ability of the Prove-it Sepsis platform, already proven as a rapid PCR- and microarray-based assay for the majority of sepsis-causing bacteria, was extended to also rapidly identify clinically relevant yeasts in blood culture. The performance characteristics of this extended platform are described. We found that the extended diagnostic Prove-it Sepsis platform was found to be highly accurate when analyzing primary isolates, spiked blood cultures, nucleic acid extracts from a retrospective blood culture data set, and primary blood cultures. Comparison of the blood culture results from the Prove-it Sepsis platform with those from conventional culture-based methods or by gene sequencing demonstrated a sensitivity of 99% and a specificity of 98% for fungal targets (based on analysis of a total of 388 specimens). Total assay time was 3 h from DNA extraction to BSI diagnosis. These results extend the performance characteristics of the Prove-it platform for bacteria to the easy, rapid, and accurate detection and species identification of yeasts in positive blood cultures. Incorporation of this extended and rapid diagnostic platform into the tools for clinical patient management would allow possibly faster identification and more focused therapies for BSIs.
Asunto(s)
Candida/clasificación , Candida/aislamiento & purificación , Candidemia/microbiología , Análisis por Micromatrices/métodos , Técnicas de Diagnóstico Molecular/métodos , Micología/métodos , Reacción en Cadena de la Polimerasa/métodos , Candida/genética , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
We present a high-throughput roll-to-roll (R2R) manufacturing process for foil-based polymethyl methacrylate (PMMA) chips of excellent optical quality. These disposable, R2R hot embossed microfluidic chips are used for the identification of the antibiotic resistance gene mecA in Staphylococcus epidermidis. R2R hot embossing is an emerging manufacturing technology for polymer microfluidic devices. It is based on continuous feeding of a thermoplastic foil through a pressurized area between a heated embossing cylinder and a blank counter cylinder. Although mass fabrication of foil-based microfluidic chips and their use for biological applications were foreseen already some years ago, no such studies have been published previously.
Asunto(s)
Proteínas Bacterianas/genética , Electroforesis en Gel de Agar , Técnicas Analíticas Microfluídicas/instrumentación , Staphylococcus epidermidis/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/análisis , Farmacorresistencia Bacteriana/genética , Técnicas Analíticas Microfluídicas/métodos , Microscopía Confocal , Polimetil Metacrilato/químicaRESUMEN
BACKGROUND: New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround time of a new molecular sepsis assay. METHODS: 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE, and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. FINDINGS: 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94.7% (95% CI 93.6-95.7) and a specificity of 98.8% (98.1-99.2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1-2 working days. 34 of 3284 (1.0%) samples were excluded because of technical and operator errors. INTERPRETATION: Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis.
Asunto(s)
Bacteriemia/microbiología , Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Bacterianas/análisis , Técnicas Bacteriológicas , Girasa de ADN/análisis , Topoisomerasa de ADN IV/análisis , Farmacorresistencia Bacteriana/genética , Genes Bacterianos , Humanos , Técnicas de Amplificación de Ácido Nucleico , Proteínas de Unión a las Penicilinas , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples. RESULTS: Comparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence. CONCLUSION: The assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Bacterias/clasificación , Bacterias/aislamiento & purificación , Girasa de ADN/genética , Cartilla de ADN , Topoisomerasa de ADN IV/genética , ADN Bacteriano/genética , Genes Bacterianos , Humanos , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Usher syndrome (USH) is an autosomal recessive disorder resulting in retinal degeneration and sensorineural deafness caused by mutations in at least 10 gene loci. USH is divided into three main clinical types: USH1 (33-44%), USH2 (56-67%), and USH3. Worldwide, USH1 and USH2 account for most of the Usher syndrome cases with rare occurrence of USH3. In Finland, however, USH3 is the most common type (40%), explained by genetic and geographical isolation accompanied with a founder mutation, while USH1 is estimated to comprise 34% and USH2 12% of all USH cases. METHODS: We examined two unrelated Finnish USH1 patients by sequencing. RESULTS: We found three new myosin VIIA (MYO7A) mutations: p.K923AfsX8, p.Q1896X, and p.E1349K. The p.K923AfsX8 mutation was present in both patients as well as in one of 200 Finnish control chromosomes. CONCLUSIONS: This is the first molecular genetic study of USH1 in Finland. We have found three new pathological mutations causing either premature termination of translation or replacement of an evolutionary conserved MYO7A amino acid.