scholarly journals Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

2013 ◽  
Vol 79 (24) ◽  
pp. 7882-7895 ◽  
Author(s):  
Kiril Tuntevski ◽  
Brandon C. Durney ◽  
Anna K. Snyder ◽  
P. Rocco LaSala ◽  
Ajay P. Nayak ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Morphological Characterization ◽  
Clinical Samples ◽  
Content Type ◽  
Bronchoalveolar Fluid ◽  
Immunosuppressed Patients ◽  
Amplicon Size ◽  
Species Specific ◽  
Ubiquitous Presence ◽  
Specific Conservation

ABSTRACTThe genusAspergillusis a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection ofAspergilluscolonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of theAspergilluscollagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of theAspergillusgenus, includingAspergillus fumigatus,A. flavus,A. nidulans,A. niger, andA. terreus. Genetic polymorphism and phylogenetic analysis of theaclF1gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination betweenAspergillusspecies. Overall, this study demonstrated thatAspergillusaclgenes could be used as PCR targets to discriminate between clinically relevantAspergillusspecies. Future studies aim to utilize the detection ofAspergillusaclgenes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification ofAspergilluscolonization and invasive aspergillosis in immunocompromised subjects.

scholarly journals Coelomycetous Fungi in the Clinical Setting: Morphological Convergence and Cryptic Diversity

2016 ◽  
Vol 55 (2) ◽  
pp. 552-567 ◽  
Author(s):  
Nicomedes Valenzuela-Lopez ◽  
Deanna A. Sutton ◽  
José F. Cano-Lira ◽  
Katihuska Paredes ◽  
Nathan Wiederhold ◽  
...  
Keyword(s):  
Large Subunit ◽  
Antifungal Susceptibility ◽  
Molecular Study ◽  
Morphological Characterization ◽  
Clinical Samples ◽  
Anatomical Site ◽  
Large Set ◽  
Content Type ◽  
Neoscytalidium Dimidiatum

ABSTRACTHuman infections by coelomycetous fungi are becoming more frequent and range from superficial to systemic dissemination. Traumatic implantation of contaminated plant material is the most common cause. The typical morphological feature of these fungi is the production of asexual spores (conidia) within fruiting bodies called conidiomata. This study aimed to determine the distribution of the coelomycetes in clinical samples by a phenotypic and molecular study of a large set of isolates received from a U.S. reference mycological institution and by obtaining thein vitroantifungal susceptibility pattern of nine antifungals against a selected group of isolates. A total of 230 isolates were identified by sequencing the D1 and D2 domains of the large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene and by morphological characterization. Eleven orders of the phylumAscomycotawere identified:Pleosporales(the largest group; 66.1%),Botryosphaeriales(19.57%),Glomerellales(4.35%),Diaporthales(3.48%),Xylariales(2.17%),HysterialesandValsariales(0.87%), andCapnodiales,Helotiales,HypocrealesandMagnaporthales(0.43% each). The most prevalent species wereNeoscytalidium dimidiatum,Paraconiothyriumspp.,Phoma herbarum,Didymella heteroderae, andEpicoccum sorghinum. The most common anatomical site of isolation was superficial tissue (66.5%), followed by the respiratory tract (17.4%). Most of the isolates tested were susceptible to the majority of antifungals, and only flucytosine showed poor antifungal activity.

scholarly journals Assembly and Annotation of Pneumocystis jirovecii from the Human Lung Microbiome

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Melanie T. Cushion ◽  
Scott P. Keely
Keyword(s):  
Bioinformatic Analysis ◽  
Pneumocystis Jirovecii ◽  
Clinical Samples ◽  
Whole Genome ◽  
Content Type ◽  
Aids Epidemic ◽  
Lung Microbiome ◽  
Immunosuppressed Patients ◽  
Novel Drug

ABSTRACTPneumocystis jiroveciiis a fungus that causesPneumocystispneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Becausein vitrocultivation ofP. jiroveciiis not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts ofP. jiroveciican be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells in clinical samples presents new challenges for whole-genome sequencing and downstream bioinformatic analysis. In a recent article, Cissé et al. used cell immunoprecipitation enrichment together with whole-genome amplification to generate sufficient quantities of DNA for Roche 454 and Illumina sequencing [O. H. Cissé, M. Pagni, and P. M. Hauser, mBio 4(1):e00428-12, 2012, doi:10.1128/mBio.00428-12]. In addition, a bioinformatic pipeline was devised to sort and assemble lung microbiome reads, thereby generating an 8.1-MbP. jiroveciigenome comprised of 356 contigs with an N50(median length of all contigs) of 41.6 kb. Knowledge of this genome will open new avenues of research, including the identification of nutritional requirements forin vitrocultivation as well as the identification of new and novel drug and vaccine targets.

scholarly journals Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens:Chlamydia psittaciandChlamydia pecorum

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3799 ◽  
Author(s):  
Martina Jelocnik ◽  
Md. Mominul Islam ◽  
Danielle Madden ◽  
Cheryl Jenkins ◽  
James Branley ◽  
...  
Keyword(s):  
Real Time ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Chlamydia Psittaci ◽  
Isothermal Amplification ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Conserved Gene ◽  
Thermal Cycler ◽  
Species Specific

BackgroundChlamydia psittaciandChlamydia pecorumare important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robustC. psittaci-specific andC. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens.Methods and MaterialsThe LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of theC. psittaci-specific Cps_0607 gene or a 209 bp region of aC. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity forC. psittaciandC. pecorumLAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples,C. psittaciLAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while forC. pecorumLAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36C. pecorumsamples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays.ResultsBoth LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min forC. psittaciandC. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% forC. psittaci(91.7% sensitivity and 92.9% specificity); and 84.1% forC. pecorum(90.6% sensitivity and 77.4% specificity). For a subset of 36C. pecorumsamples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP.DiscussionIn this study, we describe the development of novel, rapid and robustC. psittaci-specific andC. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

scholarly journals Evaluation of a Low-Density Hydrogel Microarray Technique for Mycobacterial Species Identification

2015 ◽  
Vol 53 (4) ◽  
pp. 1103-1114 ◽  
Author(s):  
Danila V. Zimenkov ◽  
Elena V. Kulagina ◽  
Olga V. Antonova ◽  
Maria A. Krasnova ◽  
Ekaterina N. Chernyaeva ◽  
...  
Keyword(s):  
Species Identification ◽  
Clinical Samples ◽  
Probe Design ◽  
Clinical Microbiology Laboratory ◽  
Low Density ◽  
Pathogenic Species ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type ◽  
Species Specific

In addition to the obligatory pathogenic species of theMycobacterium tuberculosiscomplex andMycobacterium leprae, the genusMycobacteriumalso includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of thegyrBgene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when usinggyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

scholarly journals Metagenomics of Two Severe Foodborne Outbreaks Provides Diagnostic Signatures and Signs of Coinfection Not Attainable by Traditional Methods

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Andrew D. Huang ◽  
Chengwei Luo ◽  
Angela Pena-Gonzalez ◽  
Michael R. Weigand ◽  
Cheryl L. Tarr ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pathogen Detection ◽  
Diagnostic Testing ◽  
Clinical Samples ◽  
Bioinformatics Pipeline ◽  
Shotgun Metagenomics ◽  
Content Type ◽  
Health Technologies ◽  
Culture Independent ◽  
Foodborne Outbreaks

ABSTRACT Diagnostic testing for foodborne pathogens relies on culture-based techniques that are not rapid enough for real-time disease surveillance and do not give a quantitative picture of pathogen abundance or the response of the natural microbiome. Powerful sequence-based culture-independent approaches, such as shotgun metagenomics, could sidestep these limitations and potentially reveal a pathogen-specific signature on the microbiome that would have implications not only for diagnostics but also for better understanding disease progression and pathogen ecology. However, metagenomics have not yet been validated for foodborne pathogen detection. Toward closing these gaps, we applied shotgun metagenomics to stool samples collected from two geographically isolated (Alabama and Colorado) foodborne outbreaks, where the etiologic agents were identified by culture-dependent methods as distinct strains of Salmonella enterica subsp. enterica serovar Heidelberg. Metagenomic investigations were consistent with the culture-based findings and revealed, in addition, the in situ abundance and level of intrapopulation diversity of the pathogen, the possibility of coinfections with Staphylococcus aureus, overgrowth of commensal Escherichia coli, and significant shifts in the gut microbiome during infection relative to reference healthy samples. Additionally, we designed our bioinformatics pipeline to deal with several challenges associated with the analysis of clinical samples, such as the high frequency of coeluting human DNA sequences and assessment of the virulence potential of pathogens. Comparisons of these results to those of other studies revealed that in several, but not all, cases of diarrheal outbreaks, the disease and healthy states of the gut microbial community might be distinguishable, opening new possibilities for diagnostics. IMPORTANCE Diagnostic testing for enteric pathogens has relied for decades on culture-based techniques, but a total of 38.4 million cases of foodborne illness per year cannot be attributed to specific causes. This study describes new culture-independent metagenomic approaches and the associated bioinformatics pipeline to detect and type the causative agents of microbial disease with unprecedented accuracy, opening new possibilities for the future development of health technologies and diagnostics. Our tools and approaches should be applicable to other microbial diseases in addition to foodborne diarrhea.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Screening for Intermediately Vancomycin-Susceptible and Vancomycin-Heteroresistant Staphylococcus aureus by Use of Vancomycin-Supplemented Brain Heart Infusion Agar Biplates: Defining Growth Interpretation Criteria Based on Gold Standard Confirmation

2015 ◽  
Vol 53 (11) ◽  
pp. 3543-3546 ◽  
Author(s):  
Riad Khatib ◽  
Kathleen Riederer ◽  
Mamta Sharma ◽  
Stephen Shemes ◽  
Sugantha P. Iyer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gold Standard ◽  
Population Analysis ◽  
Area Under The Curve ◽  
Brain Heart Infusion ◽  
Roc Curves ◽  
Clinical Samples ◽  
Content Type ◽  
Interpretation Criteria ◽  
Profile Area

BHI agars supplemented with vancomycin 4 (BHI-V4) and 3 (BHI-V3) mg/liter have been proposed for screening vancomycin intermediately susceptibleStaphylococcus aureus(VISA) and heteroresistant (hVISA) phenotypes, respectively, but growth interpretation criteria have not been established. We reviewed the growth results (CFU) during population analysis profile-area under the curve (PAP-AUC) of consecutive methicillin-resistantStaphylococcus aureus(MRSA) blood isolates, which were saved intermittently between 1996 and 2012. CFU counts on BHI-V4 and BHI-V3 plates were stratified according to PAP-AUC interpretive criteria: <0.90 (susceptible [S-MRSA]), 0.90 to 1.3 (hVISA), and >1.3 (VISA). CFU cutoffs that best predict VISA and hVISA were determined with the use of receiver operating characteristic (ROC) curves. Mu3, Mu50, and methicillin-susceptibleS. aureus(MSSA) controls were included. We also prospectively evaluated manufacturer-made BHI-V3/BHI-V4 biplates for screening of 2010-2012 isolates. The PAP-AUC of 616 clinical samples was consistent with S-MRSA, hVISA, and VISA in 550 (89.3%), 48 (7.8%), and 18 (2.9%) instances, respectively. For VISA screening on BHI-V4, a cutoff of 2 CFU/droplet provided 100% sensitivity and 97.7% specificity. To distinguish VISA from hVISA, a cutoff of 16 CFU provided 83.3% sensitivity and 94.7% specificity; the specificity was lowered to 89.5% with a 12-CFU cutoff. For detecting hVISA/VISA on BHI-V3, a 2-CFU/droplet cutoff provided 98.5% sensitivity and 93.8% specificity. These results suggest that 2-CFU/droplet cutoffs on BHI-V4 and BHI-V3 best approximate VISA and hVISA gold standard confirmation, respectively, with minimal overlap in samples with borderline PAP-AUC. Simultaneous screening for VISA/hVISA on manufacturer-made BHI-V4/BHI-V3 biplates is easy to standardize and may reduce the requirement for PAP-AUC confirmation.

scholarly journals Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Jean F. Challacombe ◽  
Jeannine M. Petersen ◽  
La Verne Gallegos-Graves ◽  
David Hodge ◽  
Segaran Pillai ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Response Times ◽  
Amino Acid Sequences ◽  
Clinical Samples ◽  
Whole Genome ◽  
Multiple Gene ◽  
Content Type ◽  
Environmental Surveys ◽  
Genome Comparisons

ABSTRACT Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

scholarly journals A Review of Murine Cytomegalovirus as a Model for Human Cytomegalovirus Disease—Do Mice Lie?

2020 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
Michelle A. Fisher ◽  
Megan L. Lloyd
Keyword(s):  
Human Cytomegalovirus ◽  
Murine Cytomegalovirus ◽  
Clinical Samples ◽  
Mouse Strains ◽  
Cytomegalovirus Disease ◽  
Wild Mice ◽  
Congenital Cytomegalovirus ◽  
Non Invasive ◽  
Species Specific ◽  
Disseminated Disease

Since murine cytomegalovirus (MCMV) was first described in 1954, it has been used to model human cytomegalovirus (HCMV) diseases. MCMV is a natural pathogen of mice that is present in wild mice populations and has been associated with diseases such as myocarditis. The species-specific nature of HCMV restricts most research to cell culture-based studies or to the investigation of non-invasive clinical samples, which may not be ideal for the study of disseminated disease. Initial MCMV research used a salivary gland-propagated virus administered via different routes of inoculation into a variety of mouse strains. This revealed that the genetic background of the laboratory mice affected the severity of disease and altered the extent of subsequent pathology. The advent of genetically modified mice and viruses has allowed new aspects of disease to be modeled and the opportunistic nature of HCMV infection to be confirmed. This review describes the different ways that MCMV has been used to model HCMV diseases and explores the continuing difficulty faced by researchers attempting to model HCMV congenital cytomegalovirus disease using the mouse model.

scholarly journals Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

2015 ◽  
Vol 59 (7) ◽  
pp. 4356-4359 ◽  
Author(s):  
Oliver Bader ◽  
Jana Tünnermann ◽  
Anna Dudakova ◽  
Marut Tangwattanachuleeporn ◽  
Michael Weig ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Antifungal Drug ◽  
Clinical Samples ◽  
Environmental Isolates ◽  
Northern Germany ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
The World

ABSTRACTAzole antifungal drug resistance inAspergillus fumigatusis an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

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