RESUMEN
BACKGROUND: Urine culture images collected using bacteriology automation are currently interpreted by technologists during routine standard-of-care workflows. Machine learning may be able to improve the harmonization of and assist with these interpretations. METHODS: A deep learning model, BacterioSight, was developed, trained, and tested on standard BD-Kiestra images of routine blood agar urine cultures from 2 different medical centers. RESULTS: BacterioSight displayed performance on par with standard-of-care-trained technologist interpretations. BacterioSight accuracy ranged from 97% when compared to standard-of-care (single technologist) and reached 100% when compared to a consensus reached by a group of technologists (gold standard in this study). Variability in image interpretation by trained technologists was identified and annotation "fuzziness" was quantified and found to correlate with reduced confidence in BacterioSight interpretation. Intra-testing (training and testing performed within the same institution) performed well giving Area Under the Curve (AUC) ≥0.98 for negative and positive plates, whereas, cross-testing on images (trained on one institution's images and tested on images from another institution) showed decreased performance with AUC ≥0.90 for negative and positive plates. CONCLUSIONS: Our study provides a roadmap on how BacterioSight or similar deep learning prototypes may be implemented to screen for microbial growth, flag difficult cases for multi-personnel review, or auto-verify a subset of cultures with high confidence. In addition, our results highlight image interpretation variability by trained technologist within an institution and globally across institutions. We propose a model in which deep learning can enhance patient care by identifying inherent sample annotation variability and improving personnel training.
Asunto(s)
Aprendizaje Automático , Redes Neurales de la Computación , Área Bajo la Curva , Automatización , Humanos , Flujo de TrabajoRESUMEN
Real-time PCR (RT-PCR) is widely used to diagnose human pathogens. RT-PCR data are traditionally analyzed by estimating the threshold cycle (CT ) at which the fluorescence signal produced by emission of a probe crosses a baseline level. Current models used to estimate the CT value are based on approximations that do not adequately account for the stochastic variations of the fluorescence signal that is detected during RT-PCR. Less common deviations become more apparent as the sample size increases, as is the case in the current SARS-CoV-2 pandemic. In this work, we employ a method independent of CT value to interpret RT-PCR data. In this novel approach, we built and trained a deep learning model, qPCRdeepNet, to analyze the fluorescent readings obtained during RT-PCR. We describe how this model can be deployed as a quality assurance tool to monitor result interpretation in real time. The model's performance with the TaqPath COVID19 Combo Kit assay, widely used for SARS-CoV-2 detection, is described. This model can be applied broadly for the primary interpretation of RT-PCR assays and potentially replace the CT interpretive paradigm.
Asunto(s)
COVID-19 , Aprendizaje Profundo , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
Genomic islands, defined as large clusters of genes mobilized through horizontal gene transfer, have a profound impact on evolution of prokaryotes. Recently, we developed a new program, IslandCafe, for identifying such large localized structures in bacterial genomes. A unique attribute of IslandCafe is its ability to decipher mosaic structures within genomic islands. Mosaic genomic islands have generated immense interest due to novel traits that have been attributed to such islands. To provide the Pseudomonas research community a catalogue of mosaic islands in Pseudomonas spp., we applied IslandCafe to decipher genomic islands in 224 completely sequenced genomes of Pseudomonas spp. We also performed comparative genomic analysis using BLAST to infer potential sources of distinct segments within genomic islands. Of the total 4271 genomic islands identified in Pseudomonas spp., 1036 were found to be mosaic. We also identified drug-resistant and pathogenic genomic islands and their potential donors. Our analysis provides a useful resource for Pseudomonas research community to further examine and interrogate mosaic islands in the genomes of interest and understand their role in the emergence and evolution of novel traits.
Asunto(s)
Genoma Bacteriano/genética , Islas Genómicas/genética , Pseudomonas/genética , Biología Computacional , Transferencia de Gen Horizontal , Genómica , Programas InformáticosRESUMEN
Helicobacter pylori antibiotic resistance is widespread and increasing worldwide. Routine detection of H. pylori mutations that invoke antimicrobial resistance may be a useful approach to guide antimicrobial therapy and possibly avert treatment failure. In this study, formalin-fixed, paraffin-embedded (FFPE) gastric biopsy specimens from a cohort of individuals from northern Ohio in the United States were examined using a next-generation sequencing (NGS) assay to detect H. pylori mutations that are known to confer resistance to clarithromycin, levofloxacin, and tetracycline. From January 2016 to January 2017, 133 H. pylori-infected gastric biopsy specimens were identified histologically and subsequently analyzed by NGS to detect mutations in gyrA, 23S rRNA, and 16S rRNA genes. The method successfully detected H. pylori in 126 of 133 cases (95% sensitivity). Mutations conferring resistance were present in 92 cases (73%), including 63 cases with one mutation (50%) and 29 cases with mutations in multiple genes (23%). Treatment outcomes were available in 58 cases. Sixteen of the 58 cases failed therapy (28%). Therapy failure correlated with the number of mutated genes: no failure in cases with no mutations (0/15), 19% (5/27) failure in cases with one gene mutation, and 69% (11/16) failure in cases with more than one mutated gene. Common 23S rRNA mutations (A2142G or A2413G) were present in 88% (14/16) of failed cases as opposed to in only 10% (4/42) of eradicated cases (P < 0.001). This NGS assay can be used on remnant specimens collected during standard-of-care testing to detect mutations that correlate with increased risk of treatment failure. A prospective study is needed to determine if the risk of treatment failure can be decreased by using this assay to guide antibiotic therapy.
Asunto(s)
Antibacterianos/uso terapéutico , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Niño , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Femenino , Mucosa Gástrica/patología , Genes Bacterianos/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos , Análisis de Secuencia de ADN , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
Cigarette smoking causes a vast array of diseases including cardiovascular diseases. Our laboratory focuses on investigating cigarette smoke (CS)-induced cardiovascular malfunction and the responsible mechanisms utilizing the model, c-kit-positive cardiac stem cells (CSCs). The main objective of our study is to investigate whether CS extracts (CSEs) cause impairment of CSC functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of CSC proteins and antioxidant enzymes, can modulate CSC functions and these modifications can be attenuated by ascorbate treatment. Our specific aims are (1) to investigate CSE-induced oxidative modification of CSC proteins via carbonylation, and prevention by ascorbic acid; (2) to investigate CSE-induced oxidative modification of antioxidant enzymes and ascorbic acid-mediated modulations; and (3) to investigate CSE-induced changes in CSC functions and protection by ascorbic acid. CSCs were cultured, and the aqueous extracts of CSE were prepared. CSE-induced modulations of CSC viability, oxidative modification of proteins, and antioxidant enzyme activities were detected using standard assays including Apostain, bromodeoxyuridine, and Oxiblot. CSE caused oxidative modification of CSC proteins, changed antioxidant enzyme levels, attenuated CSC proliferation, and accelerated CSC apoptosis. Ascorbic acid prevented CSE-induced CSC malfunctions, and ascorbic acid therapy might be useful in smoker CSC recipients and to condition CSCs prior to the transplant in the future. Cardiac stem cell therapy is currently undergoing in clinical trials.
Asunto(s)
Ácido Ascórbico/farmacología , Mioblastos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Humo/efectos adversos , Vitaminas/farmacología , Animales , Cardiomiopatías/etiología , Cardiomiopatías/prevención & control , Células Cultivadas , Mioblastos Cardíacos/citología , Ratas , Ratas Endogámicas F344 , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
BACKGROUND: In the model legume Medicago truncatula, the near saturation genome-wide Tnt1 insertion mutant population in ecotype R108 is a valuable tool in functional genomics studies. Forward genetic screens have identified many Tnt1 mutants defective in nodule development and symbiotic nitrogen fixation (SNF). However, progress toward identifying the causative mutations of these symbiotic mutants has been slow because of the high copy number of Tnt1 insertions in some mutant plants and inefficient recovery of flanking sequence tags (FSTs) by thermal asymmetric interlaced PCR (TAIL-PCR) and other techniques. RESULTS: Two Tnt1 symbiotic mutants, NF11217 and NF10547, with defects in nodulation and SNF were isolated during a forward genetic screen. Both TAIL-PCR and whole genome sequencing (WGS) approaches were used in attempts to find the relevant mutant genes in NF11217 and NF10547. Illumina paired-end WGS generated ~16 Gb of sequence data from a 500 bp insert library for each mutant, yielding ~40X genome coverage. Bioinformatics analysis of the sequence data identified 97 and 65 high confidence independent Tnt1 insertion loci in NF11217 and NF10547, respectively. In comparison to TAIL-PCR, WGS recovered more Tnt1 insertions. From the WGS data, we found Tnt1 insertions in the exons of the previously described PHOSPHOLIPASE C (PLC)-like and NODULE INCEPTION (NIN) genes in NF11217 and NF10547 mutants, respectively. Co-segregation analyses confirmed that the symbiotic phenotypes of NF11217 and NF10547 are tightly linked to the Tnt1 insertions in PLC-like and NIN genes, respectively. CONCLUSIONS: In this work, we demonstrate that WGS is an efficient approach for identification of causative genes underlying SNF defective phenotypes in M. truncatula Tnt1 insertion mutants obtained via forward genetic screens.
Asunto(s)
Genoma de Planta , Medicago truncatula/genética , Fijación del Nitrógeno/genética , Nodulación de la Raíz de la Planta/genética , Análisis de Secuencia de ADN/métodos , Biología Computacional , Ecotipo , Medicago truncatula/fisiología , Mutación , Reacción en Cadena de la Polimerasa , Simbiosis/genéticaRESUMEN
Mobilization of clusters of genes called genomic islands (GIs) across bacterial lineages facilitates dissemination of traits, such as, resistance against antibiotics, virulence or hypervirulence, and versatile metabolic capabilities. Robust delineation of GIs is critical to understanding bacterial evolution that has a vast impact on different life forms. Methods for identification of GIs exploit different evolutionary features or signals encoded within the genomes of bacteria, however, the current state-of-the-art in GI detection still leaves much to be desired. Here, we have taken a combinatorial approach that accounted for GI specific features such as compositional bias, aberrant phyletic pattern, and marker gene enrichment within an integrative framework to delineate GIs in bacterial genomes. Our GI prediction tool, DICEP, was assessed on simulated genomes and well-characterized bacterial genomes. DICEP compared favorably with current GI detection tools on real and synthetic datasets.
Asunto(s)
Genoma Bacteriano , Islas Genómicas , Islas Genómicas/genética , Genoma Bacteriano/genética , Bacterias/genética , Genómica/métodos , Filogenia , Programas Informáticos , Biología Computacional/métodosRESUMEN
OBJECTIVES: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant strains can be associated with increased transmissibility, more severe disease, and reduced effectiveness of treatments. To improve the availability of regional variant surveillance, we describe a variant genotyping system that is rapid, accurate, adaptable, and able to detect new low-level variants built with existing hospital infrastructure. METHODS: We used a tiered high-throughput SARS-CoV-2 screening program to characterize variants in a supraregional health system over 76 days. Combining targeted reverse transcription-polymerase chain reaction (RT-PCR) and selective sequencing, we screened SARS-CoV-2 reactive samples from all hospitals within our health care system for genotyping dominant and emerging variants. RESULTS: The median turnaround for genotyping was 2 days using the high-throughput RT-PCR-based screen, allowing us to rapidly characterize the emerging Delta variant. In our population, the Delta variant is associated with a lower cycle threshold value, lower age at infection, and increased vaccine-breakthrough cases. Detection of low-level and potentially emerging variants highlights the utility of a tiered approach. CONCLUSIONS: These findings underscore the need for fast, low-cost, high-throughput monitoring of regional viral sequences as the pandemic unfolds and the emergence of SARS-CoV-2 variants increases. Combining RT-PCR-based screening with selective sequencing allows for rapid genotyping of variants and dynamic system improvement.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Ensayos Analíticos de Alto Rendimiento , Humanos , Pandemias , SARS-CoV-2/genéticaRESUMEN
One of the evolutionary forces driving bacterial genome evolution is the acquisition of clusters of genes through horizontal gene transfer (HGT). These genomic islands may confer adaptive advantages to the recipient bacteria, such as, the ability to thwart antibiotics, become virulent or hypervirulent, or acquire novel metabolic traits. Methods for detecting genomic islands either search for markers or features typical of islands or examine anomaly in oligonucleotide composition against the genome background. The former tends to underestimate, missing islands that have the markers either lost or degraded, while the latter tends to overestimate, due to their inability to discriminate compositional atypicality arising because of HGT from those that are a consequence of other biological factors. We propose here a framework that exploits the strengths of both these approaches while bypassing the pitfalls of either. Genomic islands lacking markers are identified by their association with genomic islands with markers. This was made possible by performing marker enrichment and phyletic pattern analyses within an integrated framework of recursive segmentation and clustering. The proposed method, IslandCafe, compared favorably with frequently used methods for genomic island detection on synthetic test datasets and on a test-set of known islands from 15 well-characterized bacterial species. Furthermore, IslandCafe identified novel islands with imprints of likely horizontal acquisition.
Asunto(s)
Bacterias/genética , Biología Computacional/métodos , Genoma Bacteriano , Islas Genómicas , Genómica/métodos , Algoritmos , Elementos Transponibles de ADN , Evolución Molecular , Transferencia de Gen Horizontal , Reproducibilidad de los ResultadosRESUMEN
Staphylococcus aureus is a versatile pathogen that is capable of causing infections in both humans and animals. It can cause furuncles, septicaemia, pneumonia and endocarditis. Adaptation of S. aureus to the modern hospital environment has been facilitated, in part, by the horizontal acquisition of drug resistance genes, such as mecA gene that imparts resistance to methicillin. Horizontal acquisitions of islands of genes harbouring virulence and antibiotic resistance genes have made S. aureus resistant to commonly used antibiotics. To decipher genomic islands (GIs) in 22 hospital- and 9 community-associated methicillin-resistant S. aureus strains and classify a subset of GIs carrying virulence and resistance genes as pathogenicity and resistance islands respectively, we applied a host of methods for localizing genomic islands in prokaryotic genomes. Surprisingly, none of the frequently used GI prediction methods could perform well in delineating the resistance islands in the S. aureus genomes. Rather, a gene clustering procedure exploiting biases in codon usage for identifying horizontally transferred genes outperformed the current methods for GI detection, in particular in identifying the known islands in S. aureus including the SCCmec island that harbours the mecA resistance gene. The gene clustering approach also identified novel, as yet unreported islands, with many of these found to harbour virulence and/or resistance genes. These as yet unexplored islands may provide valuable information on the evolution of drug resistance in S. aureus.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Staphylococcus aureus Resistente a Meticilina/genética , Proteínas Bacterianas/genética , Transferencia de Gen Horizontal , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Proteínas de Unión a las Penicilinas/genética , Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as "genomic islands (GIs)." To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, "GEMINI." GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa.
RESUMEN
The mandible is the most frequently fractured bone in maxillofacial trauma, the treatment of which consists of reduction and fixation of dislocated fragments by open or closed approach. Innovative techniques toward reducing the period of the postoperative intermaxillary fixation (IMF) are being researched. A relatively unknown treatment that may have an effect on fracture healing is ultrasound. Recent clinical trials have shown that low-intensity pulsed ultrasound (LIPUS) has a positive effect on bone healing. The aim of this study was to evaluate the effect of LIPUS on healing by its application in fresh, minimally displaced or undisplaced mandibular fracture in young and healthy individuals. A total of 28 healthy patients were selected randomly from the outpatient department needing treatment of mandibular fractures. They were then randomly allocated to either of the following two groups-experimental group and study group. After IMF, patients in experimental group received pulsed ultrasound signals with frequency of 1 MHz, with temporal and spatial intensity of 1.5 W/cm(2), pulsed wave for 5 minutes on every alternate day for 24 days, whereas patients in control group received no therapy except IMF. Radiographic density at the fracture zone was assessed from the radiograph by Emago (Emago, Amsterdam, Netherlands) Image Analysis software before IMF then at 1st to 5th weeks post-IMF. The amount of clinical mobility between fracture fragments was assessed by digital manipulation of fractured fragment with the help of periodontal pocket depth measuring probe in millimeters at pre-IMF and after 3 weeks. Pain was objectively measured using a visual analogue scale at weekly interval. The data collected were subjected to unpaired "t" test. The experimental group showed significant improvement in radiographic density compared with control group at 3- and 5-week interval; pain perception was significantly reduced in experimental group compared with study group in the subsequent weeks. No significant difference was found in clinical mobility between fracture fragments at 3-week interval. The present study provides a basis for application of therapeutic controlled ultrasound as an effective treatment modality to accelerate healing of fresh, minimally displaced mandibular fracture.
RESUMEN
INTRODUCTION: Furcation perforation can have a negative impact on the prognosis of the affected tooth by compromising the attached apparatus. Hence these perforations require immediate repair. A variety of materials have been suggested for repair, of that MTA is the most promising material. The purpose of this study was to compare the ability of Gray and White MTA to seal furcation perforations using a dye extraction method under spectrophotometer. MATERIALS AND METHODS: A total of 60 permanent mandibular molars were randomly divided into four experimental groups of 15 samples each as follows: Group A: Perforation repaired with White MTA. Group B: Perforation repaired with Gray MTA. Group C: Perforation left unsealed (positive). Group D: without perforation (negative). Dye extraction was performed using full concentration nitric acid. Dye absorbance was measured at 550 nm using spectrophotometer. The data analyzed using one-way-Anova Ratio and Unpaired t-test showing statistically significance difference among the groups. RESULT: It was seen that Group D samples without perforation showed least absorbance followed by Group A (perforation repaired with White MTA) and Group B (perforation repaired with Gray MTA). Group C (perforation left unsealed) showed highest absorbance. CONCLUSION: The White and Gray Mineral Trioxide Aggregate performed similarly as a furcation perforation repair material. There was no significant difference between the Gray MTA and White MTA.