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PURPOSE: Femorotibial angle (FTA) is a convenient measure of coronal knee alignment that can be extracted from a short knee radiograph, avoiding the additional radiation exposure and specialist equipment required for full-leg radiographs. While intra- and inter-reader reproducibility from the same image has been reported, the full scan-rescan reproducibility across images, as calculated in this study, has not. METHODS: In this study, 4589 FTA measurement pairs from 2586 subjects acquired a year apart were used to estimate FTA scan-rescan reproducibility using data from the Osteoarthritis Initiative. Subjects with radiographic progression of osteoarthritis or other conditions that may cause a change in coronal knee alignment were excluded. Measurement pairs were analysed using paired-samples t $t$ tests to detect differences and compared to symptomatic changes in Western Ontario and McMaster Universities Arthritis Index scores for joint pain, stiffness and physical function to detect correlations. RESULTS: The 95% limit of agreement and the paired-samples correlation were calculated with high precision to be [-1.76°, +1.78°] and 0.938, considerably worse than the corresponding figures for intra- and inter-reader reproducibility, without relation to symptomatic or radiographic changes in knee condition. This error will weakly attenuate R 2 ${R}^{2}$ and r $r$ values from their true values in correlative studies involving FTA. The realistic maximum value for R 2 ${R}^{2}$ is 87% and for Pearson's r $r$ is 93%. CONCLUSION: The scan-rescan reproducibility in FTA is almost double the intra- and inter-reader reliability from a single scan. At almost ±2° accuracy, FTA is inappropriate for surgical use, but it is sufficiently reproducible to produce good correlations in studies predicting disease incidence and progression. LEVEL OF EVIDENCE: Level II, retrospective study.
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Aseptic loosening is the dominant failure mechanism in contemporary knee replacement surgery, but diagnostic techniques are poorly sensitive to the early stages of loosening and poorly specific in delineating aseptic cases from infections. Smart implants have been proposed as a solution, but incorporating components for sensing, powering, processing, and communication increases device cost, size, and risk; hence, minimising onboard instrumentation is desirable. In this study, two wireless, battery-free smart implants were developed that used passive biotelemetry to measure fixation at the implant-cement interface of the tibial components. The sensing system comprised of a piezoelectric transducer and coil, with the transducer affixed to the superior surface of the tibial trays of both partial (PKR) and total knee replacement (TKR) systems. Fixation was measured via pulse-echo responses elicited via a three-coil inductive link. The instrumented systems could detect loss of fixation when the implants were partially debonded (+7.1% PKA, +32.6% TKA, both p < 0.001) and fully debonded in situ (+6.3% PKA, +32.5% TKA, both p < 0.001). Measurements were robust to variations in positioning of the external reader, soft tissue, and the femoral component. With low cost and small form factor, the smart implant concept could be adopted for clinical use, particularly for generating an understanding of uncertain aseptic loosening mechanisms.
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Artroplastia do Joelho , Prótese do Joelho , Humanos , Falha de Prótese , Reoperação/métodos , Tíbia/cirurgia , Articulação do Joelho/cirurgia , Desenho de PróteseRESUMO
Achieving osseointegration is a fundamental requirement for many orthopaedic, oral, and craniofacial implants. Osseointegration typically takes three to 6 months, during which time implants are at risk of loosening. The aim of this study was to investigate whether osseointegration could be actively enhanced by delivering controllable electromechanical stimuli to the periprosthetic bone. First, the osteoconductivity of the implant surface was confirmed using an in vitro culture with murine preosteoblasts. The effects of active treatment on osseointegration were then investigated in a 21-day ex vivo model with freshly harvested cancellous bone cylinders (n = 24; Ø10 mm × 5 mm) from distal porcine femora, with comparisons to specimens treated by a distant ultrasound source and static controls. Cell viability, proliferation and distribution was evident throughout culture. Superior ongrowth of tissue onto the titanium discs during culture was observed in the actively stimulated specimens, with evidence of ten-times increased mineralisation after 7 and 14 days of culture (p < 0.05) and 2.5 times increased expression of osteopontin (p < 0.005), an adhesive protein, at 21 days. Moreover, histological analyses revealed increased bone remodelling at the implant-bone interface in the actively stimulated specimens compared to the passive controls. Active osseointegration is an exciting new approach for accelerating bone growth into and around implants.
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Periprosthetic infection is one of the most devastating complications following orthopaedic surgery. Rapid detection of an infection can change the treatment pathway and improve outcomes for the patient. In here, we propose a miniaturized lactate biosensor developed on a flexible substrate and integrated on a small-form bone implant to detect infection. The methods for lactate biosensor fabrication and integration on a bone implant are fully described within this study. The system performance was comprehensively electrochemically characterised, including with L-lactate solutions prepared in phosphate-buffered saline and culture medium, and interferents such as acetaminophen and ascorbic acid. A proof-of-concept demonstration was then conducted with ex vivo ovine femoral heads incubated with and without exposure to Staphylococcus epidermidis. The sensitivity, current density and limit-of-detection levels achieved by the biosensor were 1.25 µA mM-1, 1.51 µA.M-1.mm-2 and 66 µM, respectively. The system was insensitive to acetaminophen, while sensitivity to ascorbic acid was half that of the sensitivity to L-lactate. In the ex vivo bone model, S. epidermidis infection was detected within 5 h of implantation, while the control sample led to no change in the sensor readings. This pioneering work demonstrates a pathway to improving orthopaedic outcomes by enabling early infection diagnosis.
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Técnicas Biossensoriais , Ácido Láctico , Infecções Estafilocócicas , Staphylococcus epidermidis , Infecção da Ferida Cirúrgica , Técnicas Biossensoriais/métodos , Animais , Staphylococcus epidermidis/isolamento & purificação , Ovinos , Infecções Estafilocócicas/diagnóstico , Infecção da Ferida Cirúrgica/diagnóstico , Ácido Láctico/análise , Ácido Láctico/química , Humanos , Tecnologia sem Fio , Próteses e Implantes , Desenho de Equipamento , Infecções Relacionadas à Prótese , Enzimas Imobilizadas/química , Ortopedia , Oxigenases de Função MistaRESUMO
We describe an assay which uses broad-spectrum, conserved-site PCR paired with mass spectrometry analysis of amplicons (PCR/electrospray ionization-mass spectrometry [ESI-MS]) to detect and identify diverse bacterial and Candida species in uncultured specimens. The performance of the assay was characterized using whole-blood samples spiked with low titers of 64 bacterial species and 6 Candida species representing the breadth of coverage of the assay. The assay had an average limit of detection of 100 CFU of bacteria or Candida per milliliter of blood, and all species tested yielded limits of detection between 20 and 500 CFU per milliliter. Over 99% of all detections yielded correct identifications, whether they were obtained at concentrations well above the limit of detection or at the lowest detectable concentrations. This study demonstrates the ability of broad-spectrum PCR/ESI-MS assays to detect and identify diverse organisms in complex natural matrices that contain high levels of background DNA.
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Bactérias/isolamento & purificação , Técnicas Biossensoriais/métodos , Sangue/microbiologia , Candida/isolamento & purificação , Técnicas Microbiológicas/métodos , Bactérias/classificação , Candida/classificação , Humanos , Espectrometria de Massas/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Invasive fungal infections are a significant cause of morbidity and mortality among immunocompromised patients. Early and accurate identification of these pathogens is central to direct therapy and to improve overall outcome. PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was evaluated as a novel means for identification of fungal pathogens. Using a database grounded by 60 ATCC reference strains, a total of 394 clinical fungal isolates (264 molds and 130 yeasts) were analyzed by PCR/ESI-MS; results were compared to phenotypic identification, and discrepant results were sequence confirmed. PCR/ESI-MS identified 81.4% of molds to either the genus or species level, with concordance rates of 89.7% and 87.4%, respectively, to phenotypic identification. Likewise, PCR/ESI-MS was able to identify 98.4% of yeasts to either the genus or species level, agreeing with 100% of phenotypic results at both the genus and species level. PCR/ESI-MS performed best with Aspergillus and Candida isolates, generating species-level identification in 94.4% and 99.2% of isolates, respectively. PCR/ESI-MS is a promising new technology for broad-range detection and identification of medically important fungal pathogens that cause invasive mycoses.
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Fungos/isolamento & purificação , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Fungos/classificação , Fungos/genética , Humanos , Micoses/diagnóstico , Micoses/microbiologiaRESUMO
BACKGROUND: Knee alignment affects the development and surgical treatment of knee osteoarthritis. Automating femorotibial angle (FTA) and hip-knee-ankle angle (HKA) measurement from radiographs could improve reliability and save time. Further, if HKA could be predicted from knee-only radiographs then radiation exposure could be reduced and the need for specialist equipment and personnel avoided. The aim of this research was to assess if deep learning methods could predict FTA and HKA angle from posteroanterior (PA) knee radiographs. METHODS: Convolutional neural networks with densely connected final layers were trained to analyse PA knee radiographs from the Osteoarthritis Initiative (OAI) database. The FTA dataset with 6149 radiographs and HKA dataset with 2351 radiographs were split into training, validation, and test datasets in a 70:15:15 ratio. Separate models were developed for the prediction of FTA and HKA and their accuracy was quantified using mean squared error as loss function. Heat maps were used to identify the anatomical features within each image that most contributed to the predicted angles. RESULTS: High accuracy was achieved for both FTA (mean absolute error 0.8°) and HKA (mean absolute error 1.7°). Heat maps for both models were concentrated on the knee anatomy and could prove a valuable tool for assessing prediction reliability in clinical application. CONCLUSION: Deep learning techniques enable fast, reliable and accurate predictions of both FTA and HKA from plain knee radiographs and could lead to cost savings for healthcare providers and reduced radiation exposure for patients.
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Aprendizado Profundo , Osteoartrite do Joelho , Humanos , Tornozelo , Reprodutibilidade dos Testes , Articulação do Joelho/cirurgia , Extremidade Inferior , Osteoartrite do Joelho/cirurgia , Estudos RetrospectivosRESUMO
Introduction: Preclinical assessment of bone remodelling onto, into or around novel implant technologies is underpinned by a large live animal testing burden. The aim of this study was to explore whether a lab-based bioreactor model could provide similar insight. Method: Twelve ex vivo trabecular bone cylinders were extracted from porcine femora and were implanted with additively manufactured stochastic porous titanium implants. Half were cultured dynamically, in a bioreactor with continuous fluid flow and daily cyclic loading, and half in static well plates. Tissue ongrowth, ingrowth and remodelling around the implants were evaluated with imaging and mechanical testing. Results: For both culture conditions, scanning electron microscopy (SEM) revealed bone ongrowth; widefield, backscatter SEM, micro computed tomography scanning, and histology revealed mineralisation inside the implant pores; and histology revealed woven bone formation and bone resorption around the implant. The imaging evidence of this tissue ongrowth, ingrowth and remodelling around the implant was greater for the dynamically cultured samples, and the mechanical testing revealed that the dynamically cultured samples had approximately three times greater push-through fixation strength (p < 0.05). Discussion: Ex vivo bone models enable the analysis of tissue remodelling onto, into and around porous implants in the lab. While static culture conditions exhibited some characteristics of bony adaptation to implantation, simulating physiological conditions with a bioreactor led to an accelerated response.
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Smart implantable electronic medical devices are being developed to deliver healthcare that is more connected, personalised, and precise. Many of these implantables rely on piezoceramics for sensing, communication, energy autonomy, and biological stimulation, but the piezoceramics with the strongest piezoelectric coefficients are almost exclusively lead-based. In this article, we evaluate the electromechanical and biological characteristics of a lead-free alternative, 0.94Bi0.5Na0.5TiO3-0.06BaTiO3 (BNT-6BT), manufactured via two synthesis routes: the conventional solid-state method (PIC700) and tape casting (TC-BNT-6BT). The BNT-6BT materials exhibited soft piezoelectric properties, with d33 piezoelectric coefficients that were inferior to commonly used PZT (PIC700: 116 pC/N; TC-BNT-6BT: 121 pC/N; PZT-5A: 400 pC/N). The material may be viable as a lead-free substitute for soft PZT where moderate performance losses up to 10 dB are tolerable, such as pressure sensing and pulse-echo measurement. No short-term harmful biological effects of BNT-6BT were detected and the material was conducive to the proliferation of MC3T3-E1 murine preosteoblasts. BNT-6BT could therefore be a viable material for electroactive implants and implantable electronics without the need for hermetic sealing.
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Comércio , Próteses e Implantes , Animais , Camundongos , Íons , Comunicação , EletrônicaRESUMO
The application of molecular diagnostic methods may improve the timeliness and accuracy with which fungi are identified. A total of 76 well-characterized reference strains of clinically relevant Candida species and 61 clinical Candida isolates were tested by repetitive sequence PCR (rep-PCR) and PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and results compared against internal transcribed spacer (ITS) ribosomal RNA gene sequencing as a reference standard. Both rep-PCR and PCR/ESI-MS correctly identified 51 isolates to the species level. When method performance was evaluated based only on genospecies included in the reference libraries, both methods yielded an accuracy of 98.1%. It may be concluded that rep-PCR and PCR/ESI-MS are highly effective at identifying clinical isolates of Candida to the species level. These methods hold promise for improving the speed and accuracy of identification of Candida spp. in clinical mycology laboratories.
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Candida/classificação , Candida/isolamento & purificação , Candidíase/diagnóstico , Técnicas Microbiológicas/métodos , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Candida/química , Candida/genética , Candidíase/microbiologia , Humanos , Sequências Repetitivas de Ácido Nucleico , Sensibilidade e EspecificidadeRESUMO
Implant failure can have devastating consequences on patient outcomes following joint replacement. Time to diagnosis affects subsequent treatment success, but current diagnostics do not give early warning and lack accuracy. This research proposes an embedded ultrasound system to monitor implant fixation and temperature - a potential indicator of infection. Requiring only two implanted components: a piezoelectric transducer and a coil, pulse-echo responses are elicited via a three-coil inductive link. This passive system avoids the need for batteries, energy harvesters, and microprocessors, resulting in minimal changes to existing implant architecture. Proof-of-concept was demonstrated in vitro for a titanium plate cemented into synthetic bone, using a small embedded coil with 10 mm diameter. Gross loosening - simulated by completely debonding the implant-cement interface - was detectable with 95% confidence at up to 12 mm implantation depth. Temperature was calibrated with root mean square error of 0.19°C at 5 mm, with measurements accurate to ±1°C with 95% confidence up to 6 mm implantation depth. These data demonstrate that with only a transducer and coil implanted, it is possible to measure fixation and temperature simultaneously. This simple smart implant approach minimises the need to modify well-established implant designs, and hence could enable mass-market adoption.
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Ortopedia , Transdutores , Cimentos Ósseos , Humanos , Próteses e Implantes , Desenho de Prótese , TemperaturaRESUMO
The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.
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Técnicas Bacteriológicas/métodos , Água Doce/microbiologia , Reação em Cadeia da Polimerase/métodos , Água do Mar/microbiologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Vibrio/classificação , Vibrio/isolamento & purificação , Toxina da Cólera/genética , DNA Bacteriano/genética , República da GeórgiaRESUMO
BACKGROUND: Healthcare workers around the world are experiencing skin injury due to the extended use of personal protective equipment (PPE) during the COVID-19 pandemic. These injuries are the result of high shear stresses acting on the skin, caused by friction with the PPE. This study aims to provide a practical lubricating solution for frontline medical staff working a 4+ hours shift wearing PPE. METHODS: A literature review into skin friction and skin lubrication was conducted to identify products and substances that can reduce friction. We evaluated the lubricating performance of commercially available products in vivo using a custom-built tribometer. FINDINGS: Most lubricants provide a strong initial friction reduction, but only few products provide lubrication that lasts for four hours. The response of skin to friction is a complex interplay between the lubricating properties and durability of the film deposited on the surface and the response of skin to the lubricating substance, which include epidermal absorption, occlusion, and water retention. INTERPRETATION: Talcum powder, a petrolatum-lanolin mixture, and a coconut oil-cocoa butter-beeswax mixture showed excellent long-lasting low friction. Moisturising the skin results in excessive friction, and the use of products that are aimed at 'moisturising without leaving a non-greasy feel' should be prevented. Most investigated dressings also demonstrate excellent performance.
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Infecções por Coronavirus/complicações , Lubrificantes/uso terapêutico , Equipamento de Proteção Individual/efeitos adversos , Pneumonia Viral/complicações , Pele/lesões , Adulto , Betacoronavirus , Fenômenos Biomecânicos , COVID-19 , Fricção , Humanos , Masculino , Corpo Clínico , Pandemias , SARS-CoV-2RESUMO
We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.
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DNA Mitocondrial/química , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Composição de Bases , Bases de Dados Genéticas , Genética Forense , HumanosRESUMO
There are few diagnostic methods that readily distinguish among community-acquired methicillin (meticillin)-resistant Staphylococcus aureus strains, now frequently transmitted within hospitals. We describe a rapid and high-throughput method for bacterial profiling of staphylococcal isolates. The method couples PCR to electrospray ionization-mass spectrometry (ESI-MS) and is performed on a platform suitable for use in a diagnostic laboratory. This profiling technology produces a high-resolution genetic signature indicative of the presence of specific genetic elements that represent distinctive phenotypic features. The PCR/ESI-MS signature accurately identified genotypic determinants consistent with phenotypic traits in well-characterized reference and clinical isolates of S. aureus. Molecular identification of the antibiotic resistance genes correlated strongly with phenotypic in vitro resistance. The identification of toxin genes correlated with independent PCR analyses for the toxin genes. Finally, isolates were correctly classified into genotypic groups that correlated with genetic clonal complexes, repetitive-element-based PCR patterns, or pulsed-field gel electrophoresis types. The high-throughput PCR/ESI-MS assay should improve clinical management of staphylococcal infections.
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Técnicas de Tipagem Bacteriana/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Impressões Digitais de DNA , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Repetições Minissatélites , Fenótipo , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Estatística como AssuntoRESUMO
We describe a high-throughput assay using PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to determine the genotypes of Staphylococcus aureus isolates. The primer sets used in the PCR/ESI-MS assay were designed to amplify the same genes analyzed in multilocus sequence typing (MLST). The method was used to identify the clonal complex and USA type of each isolate and is suitable for use in a clinical or public-health setting. The method was validated using a panel of diverse isolates from the Centers for Disease Control and Prevention that were previously characterized by MLST and pulsed-field gel electrophoresis (PFGE). Clinical isolates from two geographically distinct hospitals were characterized, and the clustering results were in agreement with those for repetitive-element PCR and PFGE. The PCR/ESI-MS method enables genotyping of over 180 samples of S. aureus per day in an automated fashion.
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Técnicas de Tipagem Bacteriana/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificação , Análise por Conglomerados , Primers do DNA/genética , DNA Bacteriano/genética , Genótipo , Humanos , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Estados UnidosRESUMO
We describe a new technology for the molecular genotyping of microbes using a platform known commercially as the Ibis T5000. The technology couples multilocus polymerase chain reaction (PCR) to electrospray ionization/mass spectrometry (PCR/ESI-MS) and was developed to provide rapid, high-throughput, and precise digital analysis of either isolated colonies or original patient specimens on a platform suitable for use in hospital or reference diagnostic laboratories or public health settings. The PCR/ESI-MS method measures digital molecular signatures from microbes, enabling real-time epidemiological surveillance and outbreak investigation. This technology will facilitate understanding of the pathways by which infectious organisms spread and will enable appropriate interventions on a time frame not previously achievable.
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Infecção Hospitalar/prevenção & controle , Genética Microbiana/métodos , Epidemiologia Molecular/métodos , Vigilância da População/métodos , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Surtos de Doenças , Genes Bacterianos , Genótipo , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização por Electrospray/métodos , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
We have developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay for the rapid detection, identification, and serotyping of human adenoviruses. The assay employs a high-performance mass spectrometer to "weigh" the amplicons obtained from PCR using primers designed to amplify known human adenoviruses. Masses are converted to base compositions and, by comparison against a database of the genetic sequences, the serotype present in a sample is determined. The performance of the assay was demonstrated with quantified viral standards and environmental and human clinical samples collected from a military training facility. Over 500 samples per day can be analyzed with sensitivities greater than 100 genomes per reaction. This approach can be applied to many other families of infectious agents for rapid and sensitive analysis.
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Infecções por Adenoviridae/diagnóstico , Infecções por Adenoviridae/virologia , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Microbiologia Ambiental , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenoviridae/genética , Chlamydiales , Primers do DNA/genética , Processamento Eletrônico de Dados , Humanos , Sensibilidade e Especificidade , Sorotipagem/métodosRESUMO
In this work we report on a high-throughput mass spectrometry-based technique for the rapid high-resolution identification of Campylobacter jejuni strain types. This method readily distinguishes C. jejuni from C. coli, has a resolving power comparable to that of multilocus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high-performance mass spectrometry, which "weighs" PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the numbers of A's, G's, C's, and T's). Amplicons are derived from PCR primers which amplify short (<140-bp) regions of the housekeeping genes used by conventional MLST strategies. The results obtained with a challenge panel that comprised 25 strain types of C. jejuni and 25 strain types of C. coli are presented. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR from pure isolates.
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Campylobacter/classificação , Campylobacter/genética , Espectrometria de Massas/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Campylobacter/química , Campylobacter coli/classificação , Campylobacter coli/genética , Campylobacter jejuni/classificação , Campylobacter jejuni/genética , Primers do DNA , DNA Bacteriano/análise , Genótipo , Humanos , Especificidade da EspécieRESUMO
Newly emergent infectious diseases are a global public health problem. The population dense regions of Southeast Asia are the epicenter of many emerging diseases, as evidenced by the outbreak of Nipah, SARS, avian influenza (H5N1), Dengue, and enterovirus 71 in this region in the past decade. Rapid identification, epidemiologic surveillance, and mitigation of transmission are major challenges in ensuring public health safety. Here we describe a powerful new approach for infectious disease surveillance that is based on polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry (ESI-MS) for accurate mass measurements of the PCR products, and base composition signature analysis to identify organisms in a sample. This approach is capable of automated analysis of more than 1,500 PCR reactions a day. It is applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens and will facilitate rapid characterization of known and emerging pathogens.