Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics

Goldberg, Brittany; Sichtig, Heike; Geyer, Chelsie N.; Ledeboer, Nathan A.; Weinstock, George M.

doi:10.1128/mbio.01888-15

Cited by 289 publications

(239 citation statements)

References 30 publications

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“…Specifically, mNGS was likely more sensitive for detection of CHIKV than the CHIKV PCR used in the current study, given that 8.6% and 9.8% of the viral genome was recovered by mNGS from two CHIKV-negative samples (negative by PCR), while mNGS was less sensitive or as sensitive as the ZIKV PCR assays (Table 1). Such discrepancies between mNGS and PCR at very low viral titers have been previously reported in the other metagenomic studies (26,27) and can potentially be addressed by formal clinical validation of mNGS assay performance and the use of rigorous negative and positive controls (28).…”

Section: Discussionmentioning

confidence: 70%

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

et al. 2016

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Zika virus (ZIKV), a flavivirus, and chikungunya virus (CHIKV), an alphavirus, are infectious RNA arboviruses transmitted to humans by the bite of Aedes species mosquitoes. Both viruses have only recently emerged in the Western Hemisphere (1, 2), and along with dengue virus (DENV), another flavivirus, now circulate widely in Brazil. The acute illness caused by these viruses, characterized by fever, rash, myalgia, arthralgia, and conjunctivitis, is nonspecific, and differential diagnosis on the basis of clinical findings alone is challenging. Later infectious sequelae include chronic arthritis for CHIKV (2) and encephalitis, immune-mediated syndromes, and stroke for DENV (3). Recently, the association between ZIKV infection and severe fetal complications such as microcephaly in pregnant women has been established (4), and the virus has also been linked to neurological complications such as Guillain-Barré syndrome (5). Thus, broadbased assays are needed for differential diagnosis of vectorborne febrile illnesses and to identify potential coinfections. Here we report the utility of metagenomic next-generation sequencing (mNGS) as a screening tool to identify coinfections and the use of genome recovery and phylogenetic analyses directly from patient serum samples in the context of the ongoing ZIKV outbreak. We also show that the clinical presentation of arboviral coinfections seems to favor the virus present at a higher titer in acutely infected individuals. MATERIALS AND METHODSZIKV serum sample collection, ZIKV RT-PCR, and DENV antibody testing. Written consent from patients was obtained under a study protocol approved by the Brazil Ministry of Health (Certificado de Apresentação para Apreciação Ética 45483115.0.0000.0046, no. 1159.184, Brazil). Serum samples were obtained from 15 patients seen at Aliança Hospital in Salvador, Bahia, Brazil, from April 2015 to January 2016 who were given a presumptive diagnosis of an acute viral illness by emergency department physicians and were found to be positive by qualitative reverse transcription-PCR (RT-PCR) testing for ZIKV. Serum samples Bahia01 to Bahia15 were subjected to RNA extraction using the QIAamp viral RNA minikit (Qiagen), and RNA was reverse transcribed using the Superscript II reverse transcriptase kit (Invitrogen), followed by qualitative RT-PCR testing for ZIKV using primers targeting the NS5 gene (6). Serum samples were also tested for DENV infection using an enzyme-linked immunosorbent assay (ELISA) specific for the NS1 antigen and anti-DENV IgG/IgM according to the manufacturer's instructions (Dengue Duo Test; Bioeasy Diagnostica, Brazil).Metagenomic next-generation sequencing. A separate serum aliquot was extracted for total nucleic acid using the Qiagen viral RNA minikit (Qiagen), followed by DNase treatment using a cocktail of Turbo DNase (Thermo Fisher Scientific) and Baseline-Zero DNase (Epicentre Biotechnologies), followed by NGS library construction using the NexteraXT kit (Illumina) as previously described (7,8). Runs of single-end, 160-base pair...

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Section: Discussionmentioning

confidence: 70%

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

et al. 2016

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“…It is also gaining interest in the field of clinical microbiology as a way to identify pathogens in normally sterile specimens (1)(2)(3)(4)(5)(6)(7). Not only does this approach allow for pathogen identification, but the gene content information can also be used for other analyses, such as antibiotic resistance prediction (8,9), typing for tracking of outbreaks or epidemiological studies (10), or assessment for other relevant genes, such as those encoding virulence factors (11,12).…”

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confidence: 99%

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

et al. 2017

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Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.KEYWORDS metagenomics, prosthetic joint infection, whole-genome amplification M etagenomic shotgun sequencing is a rapidly developing powerful tool to examine microbial communities. It is also gaining interest in the field of clinical microbiology as a way to identify pathogens in normally sterile specimens (1-7). Not only does this approach allow for pathogen identification, but the gene content information can also be used for other analyses, such as antibiotic resistance prediction (8, 9), typing for tracking of outbreaks or epidemiological studies (10), or assessment for other relevant genes, such as those encoding virulence factors (11,12).One significant barrier to metagenomic shotgun sequencing approaches is the amount of genetic material necessary to perform these experiments. This is not an obstacle in settings such as fecal microbiome studies, where abundant organisms are present, but in other scenarios (e.g., in scenarios with low-biomass environmental samples and uncultured bacteria or in single cell analysis), the number of cells and the amount of genetic material can be limited (13)(14)(15)(16)(17). It is in these situations that whole-genome amplification (WGA) has the potential to play an important role in pathogen detection. Citation Thoendel M, Jeraldo P, GreenwoodQuaintance KE, Yao J, Chia N, Hanssen AD, Abdel MP, Patel R. 2017. Impact of contaminating DNA in whole-genome amplification kits used for metagenomic shotgun sequencing for infection diagnosis. J

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“…One of the recommendations from the colloquium report is that clinical microbiologists and other relevant stakeholders should work with representatives from organizations, including the FDA, Centers for Medicare and Medicaid Services (CMS), NIH, CDC, and CAP, to develop specific guidelines for the validation of NGS-based diagnostic assays (12,30). There is a need for pathogen-specific guidance for the validation and QC procedures unique to the variety of etiological agents that can be detected using NGS (e.g., bacteria, viruses, fungi, yeasts, and parasites).…”

Section: Discussionmentioning

confidence: 99%

“…No clinical microbiology NGS tests have been approved by the FDA, and the limited number of clinical infectious disease NGS-based assays currently offered are being performed as LDTs. The FDA is beginning to evaluate the critical components for the clearance/approval of infectious disease NGS-based assays, particularly for pathogen identification and detection of antimicrobial resistance markers, and it has published both a discussion paper and draft guidance describing the current considerations for the approaches for approval/clearance of NGS diagnostic devices for clinical microbiology (23,30,36).…”

Section: Discussionmentioning

confidence: 99%

“…The strains chosen are relevant to food safety and clinical microbiology NGS applications and represent diverse genome sizes, plasmid contents, and GC contents (10). Other RMs that may be useful for NGS for validation and evaluation of the bioinformatics pipeline include synthetic DNA samples, such as plasmids containing known variants that can be engineered to represent a broad range of sequences and variant types or synthetic "armored RNA" capsids that can be used to simulate infectious viral particles spiked into clinical matrices; and electronic or digital reference materials, such as curated benchmark data sets (i.e., well-characterized and complete genome data sets derived from a variety of organisms relevant to clinical and public health microbiology) (10,11,16,30).…”

Section: Considerations For Ngs Reference Materials and Proficiency Tmentioning

confidence: 99%

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Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories

2016

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Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials. N ext-generation sequencing (NGS) is transforming the landscape of clinical microbiology and public health laboratories. The applications of NGS are wide-ranging and include wholegenome sequencing, microbiome analysis/metagenomics, transcriptome profiling, infectious disease diagnosis, pathogen discovery, and public health surveillance. For example, NGS has recently been used to better understand hospital outbreaks and inform infection control practices (1), and it can be used in the clinical microbiology laboratory to identify unknown organisms, predict antimicrobial resistance, assess virulence gene content, and inform molecular epidemiology efforts (2). Metagenomic "unbiased" NGS applications, coupled with recently developed bioinformatics solutions (3-5) that enable the identification of all pathogens directly from a clinical sample based on sequence homology, have the potential to complement or even replace current standard clinical laboratory tests. For example, the use of metagenomics combined with a rapid bioinformatics pipeline recently facilitated a clinically actionable diagnosis of neuroleptospirosis when conventional testing was initially unable to identify the causative organism (6). A number of agencies are working to bring NGS into the public health laboratory setting. For example, through the U.S. Centers for Disease Control and Prevention (CDC) Advanced Molecular Detection (AMD) Initiative, national, state, and local partners are beginning to incorporate NGSbased methods into disease surveillance systems. AMD initiatives include broad applications of NGS to address public health problems, including vaccine improvement, identification of emerging threats, and tracking diseases and outbreaks (http://www.cdc.gov /amd/). The CDC, Food and Drug Administration (FDA), National Institutes of Health (NIH), National Center for Biotechnology Information (NCBI), National Library of Medicine, and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) have established an Interagency Collaboration on Genomics and Food Safety (Gen-FS), with the goal of fostering timely access to genomic data for foodborne pathogen surveillance and ou...

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Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics

Cited by 289 publications

References 30 publications

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories

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