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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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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
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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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
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
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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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
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
UNLABELLED: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. DISCLAIMER: The IRIDICA BAC BSI Assay is not available in the United States.
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Bactérias/isolamento & purificação , Infecções Bacterianas/sangue , Bioensaio/métodos , Candida/isolamento & purificação , Candidíase/sangue , Sepse/sangue , Algoritmos , Antibacterianos/uso terapêutico , Primers do DNA , Farmacorresistência Bacteriana , Farmacorresistência Fúngica , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sepse/microbiologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Diverse viruses often reactivate in or infect cancer patients, patients with immunocompromising infections or genetic conditions, and transplant recipients undergoing immunosuppressive therapy. These infections can disseminate, leading to death, transplant rejection, and other severe outcomes. OBJECTIVES: To develop and characterize an assay capable of inclusive and accurate identification of diverse potentially disseminating viruses directly from plasma specimens. STUDY DESIGN: We developed a PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to simultaneously detect and identify adenovirus, enterovirus, polyomaviruses JC and BK, parvovirus B19, HSV-1, HSV-2, VZV, EBV, CMV, and herpesviruses 6-8 in plasma specimens. The assay performance was characterized analytically, and the results from clinical plasma samples were compared to the results obtained from single-analyte real time PCR tests currently used in clinical practice. RESULTS: The assay demonstrated sensitivity and specificity to diverse strains of the targeted viral families and robustness to interfering substances and potentially cross reacting organisms. The assay yielded 94% sensitivity when testing clinical plasma samples previously identified as positive using standard-of-care real-time PCR tests for a single target virus (available samples included positive samples for 11 viruses targeted by the assay). CONCLUSIONS: The assay functioned as designed, providing simultaneous broad-spectrum detection and identification of diverse agents of disseminated viral infection. Among 156 clinical samples tested, 37 detections were made in addition to the detections matching the initial clinical positive results.
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Patologia Molecular/métodos , Viremia/diagnóstico , Viremia/virologia , Virologia/métodos , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Vírus/classificação , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
A set of 711 samples from four U.S. population groups was analyzed using a novel mass spectrometry based method for mitochondrial DNA (mtDNA) base composition profiling. Comparison of the mass spectrometry results with Sanger sequencing derived data yielded a concordance rate of 99.97%. Length heteroplasmy was identified in 46% of samples and point heteroplasmy was observed in 6.6% of samples in the combined mass spectral and Sanger data set. Using discrimination capacity as a metric, Sanger sequencing of the full control region had the highest discriminatory power, followed by the mass spectrometry base composition method, which was more discriminating than Sanger sequencing of just the hypervariable regions. This trend is in agreement with the number of nucleotides covered by each of the three assays.
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Composição de Bases , DNA Mitocondrial/genética , Grupos Populacionais , Análise de Sequência de DNA , Sequência de Bases , Primers do DNA , Humanos , Espectrometria de Massas , Estados UnidosRESUMO
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
Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
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Bactérias/genética , Armas Biológicas , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Vírus/genética , Bactérias/isolamento & purificação , Bioensaio , Análise por Conglomerados , Primers do DNA/metabolismo , Reações Falso-Negativas , Limite de Detecção , Relatório de Pesquisa , Sensibilidade e Especificidade , Estatística como Assunto , Vírus/isolamento & purificaçãoRESUMO
Short tandem repeats (STRs) are the primary genetic markers used for the analysis of biological samples in forensic and human identity testing. The discrimination power of a combination of STRs is sufficient in many human identity testing comparisons unless the evidence is substantially compromised and/or there are insufficient relatives or a potential mutation may have arisen in kinship analyses. An automated STR assay system that is based on electrospray ionization mass spectrometry (ESI-MS) has been developed that can increase the discrimination power of some of the CODIS core STR loci and thus provide more information in typical and challenged samples and cases. Data from the ESI-MS STR system is fully backwards compatible with existing STR typing results generated by capillary electrophoresis. In contrast, however, the ESI-MS analytical system also reveals nucleotide polymorphisms residing within the STR alleles. The presence of these polymorphisms expands the number of alleles at a locus. Population studies were performed on the 13 core CODIS STR loci from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as nucleotide variations contained within repeat motifs or flanking regions. Such additional polymorphisms were identified in 11 of the 13 loci examined whereby several nominal length alleles were subdivided. A substantial increase in heterozygosity was observed, with close to or greater than 5% of samples analyzed being heterozygous with equal-length alleles in at least one of five of the core CODIS loci. This additional polymorphism increases discrimination power significantly, whereby the seven most polymorphic STR loci have a discrimination power equivalent to the 10 most discriminating of the CODIS core loci. An analysis of substructure among the three population groups revealed a higher θ than would be observed compared with using alleles designated by nominal length, i.e., repeats solely. Two loci, D3S1358 and vWA produced θ estimates of 0.0477 and 0.0234, respectively, when the expanded allele complement (i.e., nominal allele and SNPs) was considered compared to 0.0145 and 0.01266, respectively when only nominal repeat number was considered. These differences may indicate underlying population specific allele distributions exist within these populations. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.
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Alelos , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Espectrometria de Massas por Ionização por Electrospray/métodos , Automação , Sequência de Bases , Primers do DNA , Eletroforese Capilar , Humanos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Homologia de Sequência do Ácido NucleicoRESUMO
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
Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.
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Bacteriemia/diagnóstico , DNA Bacteriano/sangue , DNA Fúngico/sangue , Fungemia/diagnóstico , Técnicas de Diagnóstico Molecular , Idoso , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto JovemRESUMO
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.