The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense

Lesho, Emil; Clifford, Robert; Onmus-Leone, Fatma; Appalla, Lakshmi; Snesrud, Erik; Kwak, Yoon I.; Ong, Ana; Maybank, Rosslyn; Waterman, Paige; Rohrbeck, Patricia; Julius, Michael; Roth, Amanda L.; Martínez, Julián; Nielsen, Lindsey; Steele, Eric; McGann, Patrick; Hinkle, Mary

doi:10.1371/journal.pone.0155770

Cited by 25 publications

(30 citation statements)

References 20 publications

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“…These results strengthen the concept of unified laboratory standards for WGS enunciated by some professional organizations, including the Global Microbial Identifier (GMI) initiative [30, 31, 33, 47]. A few other groups have also highlighted the challenges and solutions for the implementation of WGS in clinical and public health microbiology laboratories [21, 48].…”

Section: Discussionsupporting

confidence: 65%

“…However, these resource-intensive networks focus on few selected pathogens at present. Notably, there still remain significant challenges for the implementation of the comprehensive WGS services at the local level [48, 57]. It is hoped that the quality framework proposed in the present study would advance the localization of comprehensive WGS services in clinical and public health laboratories.…”

Section: Discussionmentioning

confidence: 99%

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Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

Truong²,

Greninger³

et al. 2017

Preprint

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BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.Objectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.MethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.ResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.ConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

Truong²,

Greninger³

et al. 2017

Preprint

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“…Presently, the US screening efforts for multidrug-resistant bacteria were intensified and amended by whole genome next-generation sequencing. With multiple global sampling sites, the resulting strain collection comprises several 10,000 isolates [ 41 ].…”

Section: Experience From Military Medical Healthcare Facilities Inmentioning

confidence: 99%

Resistant Gram-Negative Bacteria and Diagnostic Point-of-Care Options for the Field Setting during Military Operations

Frickmann

Podbielski²,

Kreikemeyer³

2018

BioMed Research International

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The spread of multidrug-resistant bacteria in resource-poor settings affects the military medical service in case of deployments of soldiers to war and crisis zones. Patients with war injuries are prone to colonization or infection with multidrug-resistant bacteria. Resistant Gram-negative bacteria play a dominant role in military wound infections. Problematic hygiene conditions on deployment facilitate exposition of soldiers with subsequent colonization. Although colonizing strains are frequently cleared from their hosts after returning from deployment, transmission to close contacts of the soldiers in the home country cannot be excluded and therapeutic options are reduced if colonization progresses to invasive infection. Since sophisticated culture-based diagnostic approaches are typically not available in the field setting on deployment, molecular rapid diagnostic test systems are an option for transmission control if the locally prevalent molecular resistance mechanisms are known. Efforts for global resistance surveillance can contribute to better understanding of resistance distribution and spread at deployment sites. This review summarizes experience of the military medical services with multidrug resistance on deployment and with the influx of resistant strains to the home country and discusses potential use of available molecular rapid test systems as an option for the field setting.

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“…The development of smaller and less expensive gadgets is another requirement if sequencing should enter routine diagnostics. Compared with research laboratories, clinical routine laboratories rarely have staff skilled enough to prepare high-quality DNA libraries [ 55 ]. Educational and training enterprises or automatic library preparation may be solutions to this problem [ 56 ].…”

Section: Limitation Of Single Cell Sequencingmentioning

confidence: 99%

“… The sequencing of large volumes create extraordinary burdens in terms of the sharing and storage of data. Possible mitigation includes the use of FASTA/FASTQ to email sequences and cloud-based solutions [ 55 ]. …”

Section: Limitation Of Single Cell Sequencingmentioning

confidence: 99%

Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance

2016

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Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients.

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The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense

Cited by 25 publications

References 20 publications

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

Resistant Gram-Negative Bacteria and Diagnostic Point-of-Care Options for the Field Setting during Military Operations

Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance

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