scholarly journals A Combination of Metagenomic and Cultivation Approaches Reveals Hypermutator Phenotypes within Vibrio cholerae-Infected Patients

mSystems ◽  
2021 ◽  
Author(s):  
Inès Levade ◽  
Ashraful I. Khan ◽  
Fahima Chowdhury ◽  
Stephen B. Calderwood ◽  
Edward T. Ryan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Vibrio Cholerae ◽  
Genome Sequencing ◽  
Diarrheal Disease ◽  
Asymptomatic Infection ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Content Type ◽  
Life Threatening

Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers.

scholarly journals A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients

2020 ◽  
Author(s):  
Inès Levade ◽  
Ashraful I. Khan ◽  
Fahima Chowdhury ◽  
Stephen B. Calderwood ◽  
Edward T. Ryan ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Whole Genome Sequencing ◽  
Vibrio Cholerae ◽  
Genome Sequencing ◽  
Asymptomatic Infection ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Asymptomatic Carriers ◽  
Adaptive Mutations ◽  
Neutral Mutations

ABSTRACTVibrio cholerae can cause a range of symptoms in infected patients, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient have shown that Vibrio cholerae can evolve a modest amount of genetic diversity during symptomatic infection. Little is known about V. cholerae genetic diversity within asymptomatic infected patients. To achieve increased resolution in the detection of Vibrio cholerae diversity within individual infections, we applied culture-based population genomics and metagenomics to a cohort of symptomatic and asymptomatic cholera patients. While the metagenomic approach allowed us to detect more mutations in symptomatic patients compared to the culture-based approach, WGS of isolates was still necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low Vibrio cholerae load. We found that symptomatic and asymptomatic patients contain similar levels of within-patient diversity, and discovered V. cholerae hypermutators in some patients. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infection and asymptomatic infection, while providing evidence for hypermutator phenotypes within cholera patients.IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the acuteness of cholera infections, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within 6 out of 14 patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

scholarly journals Evaluation of Whole-Genome Sequencing for Identification and Typing of Vibrio cholerae

2018 ◽  
Vol 56 (11) ◽  
Author(s):  
David R. Greig ◽  
Ulf Schaefer ◽  
Sophie Octavia ◽  
Ebony Hunter ◽  
Marie A. Chattaway ◽  
...  
Keyword(s):  
Public Health ◽  
Whole Genome Sequencing ◽  
Vibrio Cholerae ◽  
Species Identification ◽  
Genome Sequencing ◽  
National Level ◽  
Whole Genome ◽  
Content Type ◽  
El Tor ◽  
The Impact

ABSTRACT Epidemiological and microbiological data on Vibrio cholerae strains isolated between April 2004 and March 2018 (n = 836) and held at the Public Health England culture archive were reviewed. The traditional biochemical species identification and serological typing results were compared with the genome-derived species identification and serotype for a subset of isolates (n = 152). Of the 836 isolates, 750 (89.7%) were from a fecal specimen, 206 (24.6%) belonged to serogroup O1, and 7 (0.8%) were serogroup O139; 792 (94.7%) isolates were from patients reporting recent travel abroad, most commonly to India (n = 209) and Pakistan (n = 104). Of the 152 V. cholerae isolates identified by use of kmer, 149 (98.1%) were concordant with those identified using the traditional biochemical approach. Traditional serotyping results were 100% concordant with those of the whole-genome sequencing (WGS) analysis for the identification of serogroups O1 and O139 and classical and El Tor biotypes. ctxA was detected in all isolates of V. cholerae O1 El Tor and O139 belonging to sequence type 69 (ST69) and in V. cholerae O1 classical variants belonging to ST73. A phylogeny of isolates belonging to ST69 from U.K. travelers clustered geographically, with isolates from India and Pakistan located on separate branches. Moving forward, WGS data from U.K. travelers will contribute to global surveillance programs and the monitoring of emerging threats to public health and the global dissemination of pathogenic lineages. At the national level, these WGS data will inform the timely reinforcement of direct public health messaging to travelers and mitigate the impact of imported infections and the associated risks to public health.

scholarly journals Serotype Diversity and Antimicrobial Resistance amongSalmonella entericaIsolates from Patients at an Equine Referral Hospital

2018 ◽  
Vol 84 (13) ◽  
pp. e02829-17 ◽  
Author(s):  
I. M. Leon ◽  
S. D. Lawhon ◽  
K. N. Norman ◽  
D. S. Threadgill ◽  
N. Ohta ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Referral Hospital ◽  
Whole Genome ◽  
Content Type ◽  
Genes Encoding ◽  
Life Threatening ◽  
Common Serotypes

ABSTRACTAlthoughSalmonella entericacan produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) ofSalmonellaisolates from patients at an equine referral hospital in the southern United States. A total of 255Salmonellaisolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes wereSalmonella entericaserotype Newport (18%),Salmonella entericaserotype Anatum (15.2%), andSalmonella entericaserotype Braenderup (11.8%). Most (n= 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such asblaCMY-2,blaSHV-12,blaCTX-M-27, andblaTEM-1B, were detected. TheqnrB2 andaac(6′)-Ib-crgenes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3′)-Ia,aac(6′)-IIc), streptomycin (strA andstrB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) andtet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMRSalmonellain equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology ofSalmonellain horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCEIn horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treatSalmonellainfections. In this study, we identify temporal trends in the distribution ofSalmonellaserotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation ofSalmonellainfections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

scholarly journals Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

2017 ◽  
Vol 55 (6) ◽  
pp. 1871-1882 ◽  
Author(s):  
Joseph Shea ◽  
Tanya A. Halse ◽  
Pascal Lapierre ◽  
Matthew Shudt ◽  
Donna Kohlerschmidt ◽  
...  
Keyword(s):  
New York ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Predictive Value ◽  
New York State ◽  
Whole Genome ◽  
Content Type ◽  
York State

ABSTRACTWhole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterizeMycobacterium tuberculosisand otherM. tuberculosiscomplex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

scholarly journals Independent Microevolution Mediated by Mobile Genetic Elements of IndividualClostridium difficileIsolates from Clade 4 Revealed by Whole-Genome Sequencing

mSystems ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Yuan Wu ◽  
Chen Liu ◽  
Wen-Ge Li ◽  
Jun-Li Xu ◽  
Wen-Zhu Zhang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clostridium Difficile ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Mobile Genetic Elements ◽  
High Rate ◽  
Whole Genome ◽  
Content Type ◽  
Genetic Elements ◽  
Sequence Types

ABSTRACTHorizontal gene transfer of mobile genetic elements (MGEs) accounts for the mosaic genome ofClostridium difficile, leading to acquisition of new phenotypes, including drug resistance and reconstruction of the genomes. MGEs were analyzed according to the whole-genome sequences of 37C. difficileisolates with a variety of sequence types (STs) within clade 4 from China. Great diversity was found in each transposon even within isolates with the same ST. Two novel transposons were identified in isolates ZR9 and ZR18, of which approximately one third to half of the genes showed heterogenous origins compared with the usual intestinal bacterial genes. Most importantly,catD, known to be harbored by Tn4453a/b, was replaced byaac(6′) aph(2′′)in isolates 2, 7, and 28. This phenomenon illustrated the frequent occurrence of gene exchanges betweenC. difficileand other enterobacteria with individual heterogeneity. Numerous prophages and CRISPR arrays were identified inC. difficileisolates of clade 4. Approximately 20% of spacers were located in prophage-carried CRISPR arrays, providing a new method for typing and tracing the origins of closely related isolates, as well as in-depth studies of the mechanism underlying genome remodeling. The rates of drug resistance were obviously higher than those reported previously around the world, although all isolates retained high sensitivity to vancomycin and metronidazole. The increasing number ofC. difficileisolates resistant to all antibiotics tested here suggests the ease with which resistance is acquiredin vivo. This study gives insights into the genetic mechanism of microevolution within clade 4.IMPORTANCEMobile genetic elements play a key role in the continuing evolution ofClostridium difficile, resulting in the emergence of new phenotypes for individual isolates. On the basis of whole-genome sequencing analysis, we comprehensively explored transposons, CRISPR, prophage, and genetic sites for drug resistance within clade 4C. difficileisolates with different sequence types. Great diversity in MGEs and a high rate of multidrug resistance were found within this clade, including new transposons, Tn4453a/bwithaac(6′) aph(2′′)instead ofcatD, and a relatively high rate of prophage-carried CRISPR arrays. These findings provide important new insights into the mechanism of genome remodeling within clade 4 and offer a new method for typing and tracing the origins of closely related isolates.

scholarly journals Whole-Genome Sequencing for Drug Resistance Profile Prediction inMycobacterium tuberculosis

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Sebastian M. Gygli ◽  
Peter M. Keller ◽  
Marie Ballif ◽  
Nicolas Blöchliger ◽  
Rico Hömke ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Critical Concentration ◽  
Drug Susceptibility Testing ◽  
Quality Data ◽  
Whole Genome ◽  
Resistance Mutations ◽  
Content Type ◽  
Agar Dilution

ABSTRACTWhole-genome sequencing allows rapid detection of drug-resistantMycobacterium tuberculosisisolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinicalM. tuberculosisisolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

scholarly journals Genomically Informed Surveillance for Carbapenem-Resistant Enterobacteriaceae in a Health Care System

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Nicole D. Pecora ◽  
Ning Li ◽  
Marc Allard ◽  
Cong Li ◽  
Esperanza Albano ◽  
...  
Keyword(s):  
Health Care ◽  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
Enterobacter Cloacae ◽  
Mobile Elements ◽  
Whole Genome ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health concern. Rapid identification of the resistance genes, their mobilization capacity, and strains carrying them is essential to direct hospital resources to prevent spread and improve patient outcomes. Whole-genome sequencing allows refined tracking of both chromosomal traits and associated mobile genetic elements that harbor resistance genes. To enhance surveillance of CREs, clinical isolates with phenotypic resistance to carbapenem antibiotics underwent whole-genome sequencing. Analysis of 41 isolates of Klebsiella pneumoniae and Enterobacter cloacae, collected over a 3-year period, identified K. pneumoniae carbapenemase (KPC) genes encoding KPC-2, −3, and −4 and OXA-48 carbapenemases. All occurred within transposons, including multiple Tn4401 transposon isoforms, embedded within more than 10 distinct plasmids representing incompatibility (Inc) groups IncR, -N, -A/C, -H, and -X. Using short-read sequencing, draft maps were generated of new KPC-carrying vectors, several of which were derivatives of the IncN plasmid pBK31551. Two strains also had Tn4401 chromosomal insertions. Integrated analyses of plasmid profiles and chromosomal single-nucleotide polymorphism (SNP) profiles refined the strain patterns and provided a baseline hospital mobilome to facilitate analysis of new isolates. When incorporated with patient epidemiological data, the findings identified limited outbreaks against a broader 3-year period of sporadic external entry of many different strains and resistance vectors into the hospital. These findings highlight the utility of genomic analyses in internal and external surveillance efforts to stem the transmission of drug-resistant strains within and across health care institutions. IMPORTANCE We demonstrate how detection of resistance genes within mobile elements and resistance-carrying strains furthers active surveillance efforts for drug resistance. Whole-genome sequencing is increasingly available in hospital laboratories and provides a powerful and nuanced means to define the local landscape of drug resistance. In this study, isolates of Klebsiella pneumoniae and Enterobacter cloacae with resistance to carbapenem antibiotics were sequenced. Multiple carbapenemase genes were identified that resided in distinct transposons and plasmids. This mobilome, or population of mobile elements capable of mobilizing drug resistance, further highlighted the degree of strain heterogeneity while providing a detailed timeline of carbapenemase entry into the hospital over a 3-year period. These surveillance efforts support effective targeting of infection control resources and the development of institution-specific repositories of resistance genes and the mobile elements that carry them.

scholarly journals Whole-Genome Sequencing of Four Campylobacter Strains Isolated from Gull Excreta Collected from Hobie Beach (Oxnard, CA, USA)

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Vicente Gomez-Alvarez ◽  
Nicholas J. Ashbolt ◽  
John F. Griffith ◽  
Jorge Santo Domingo ◽  
Jingrang Lu
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Diarrheal Disease ◽  
Draft Genome ◽  
Avian Species ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type

Campylobacter spp. are commensal organisms in avian species and are one of the leading causes of bacterial foodborne human diarrheal disease worldwide. We report the draft genome sequences of Campylobacter volucris, C. lari, and C. jejuni strains isolated from California gull (Larus californicus) excreta collected from a California beach.

scholarly journals In SilicoSerotyping Based on Whole-Genome Sequencing Improves the Accuracy ofShigellaIdentification

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Yun Wu ◽  
Henry K. Lau ◽  
Teresa Lee ◽  
David K. Lau ◽  
Justin Payne
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Diarrheal Disease ◽  
Cross Reactivity ◽  
Reference Sequence ◽  
Molecular Diagnostic ◽  
Whole Genome ◽  
Gene Markers ◽  
Content Type

ABSTRACTBacteria of the genusShigella, consisting of 4 species and >50 serotypes, cause shigellosis, a foodborne disease of significant morbidity, mortality, and economic loss worldwide. ClassicalShigellaidentification based on selective media and serology is tedious, time-consuming, expensive, and not always accurate. A molecular diagnostic assay does not distinguishShigellaat the species level or from enteroinvasiveEscherichia coli(EIEC). We inspected genomic sequences from 221Shigellaisolates and observed low concordance rates between conventional designation and molecular serotyping: 86.4% and 80.5% at the species and serotype levels, respectively. Serotype determinants for 6 additional serotypes were identified. Examination of differentiation gene markers commonly perceived as characteristic hallmarks inShigellashowed high variability among different serotypes. Using this information, we developed ShigaTyper, an automated workflow that utilizes limited computational resources to accurately and rapidly determine 59Shigellaserotypes using Illumina paired-end whole-genome sequencing (WGS) reads.Shigellaserotype determinants and species-specific diagnostic markers were first identified through read alignment to an in-house curated reference sequence database. Relying on sequence hits that passed a threshold level of coverage and accuracy, serotype could be unambiguously predicted within 1 min for an average-size WGS sample of ∼500 MB. Validation with WGS data from 380 isolates showed an accuracy rate of 98.2%. This pipeline is the first step toward building a comprehensive WGS-based analysis pipeline ofShigellaspp. in a field laboratory setting, where speed is essential and resources need to be more cost-effectively dedicated.IMPORTANCEShigellacauses diarrheal disease with serious public health implications. However, conventionalShigellaidentification methods are laborious and time-consuming and can be erroneous due to the high similarity betweenShigellaand enteroinvasiveEscherichia coli(EIEC) and cross-reactivity between serotyping antisera. Further, serotype interpretation is complicated for inexperienced users. To develop an easier method with higher accuracy based on whole-genome sequencing (WGS) forShigellaserotyping, we systematically examined genomic information ofShigellaisolates from 53 serotypes to define rules for differentiation and serotyping. We created ShigaTyper, an automated pipeline that accurately and rapidly excludes non-Shigellaisolates and identifies 59Shigellaserotypes using Illumina paired-end WGS reads. A serotype can be unambiguously predicted at a data processing speed of 538 MB/min with 98.2% accuracy from a regular laptop. Once it is installed, training in bioinformatics analysis andShigellagenetics is not required. This pipeline is particularly useful to general microbiologists in field laboratories.

scholarly journals Deciphering Within-Host Microevolution ofMycobacterium tuberculosisthrough Whole-Genome Sequencing: the Phenotypic Impact and Way Forward

2019 ◽  
Vol 83 (2) ◽  
Author(s):  
S. D. Ley ◽  
M. de Vos ◽  
A. Van Rie ◽  
R. M. Warren
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Variants ◽  
Direct Consequence ◽  
Whole Genome ◽  
Limited Data ◽  
Content Type ◽  
Sequencing Studies ◽  
Functional Relevance

SUMMARYTheMycobacterium tuberculosisgenome is more heterogenous and less genetically stable within the host than previously thought. Currently, only limited data exist on the within-host microevolution, diversity, and genetic stability ofM. tuberculosis. As a direct consequence, our ability to inferM. tuberculosistransmission chains and to understand the full complexity of drug resistance profiles in individual patients is limited. Furthermore, apart from the acquisition of certain drug resistance-conferring mutations, our knowledge on the function of genetic variants that emerge within a host and their phenotypic impact remains scarce. We performed a systematic literature review of whole-genome sequencing studies of serial and parallel isolates to summarize the knowledge on genetic diversity and within-host microevolution ofM. tuberculosis. We identified genomic loci of within-host emerged variants found across multiple studies and determined their functional relevance. We discuss important remaining knowledge gaps and finally make suggestions on the way forward.

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