Next-generation sequencing in clinical microbiology

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a series of machines and protocols capable of changing the practice of microbiology for the diagnosis of infectious disease [1]. These are continually compared ...
Editorial

Next-generation sequencing in clinical microbiology Expert Rev. Mol. Diagn. 13(3), 00–00 (2013)

“In terms of monitoring resistance, next-generation sequencing

Eleni Mavrogiorgou

Technological developments in the generation of nucleic acid sequence data, collectively known as next-generation sequencing (NGS), have provided us with a series of machines and protocols capable of changing the practice of microbiology for the diagnosis of infectious disease [1] . These are continually compared and technical updates are regularly published from both the general clinical [2] and specific microbial perspectives [3–5] . The pace of change in the technology however means that the relevance of such comparisons is questioned and little impact has been seen to date on the management of patients with an infectious disease. It is very difficult for busy clinical and public health professionals to keep up with the latest information on NGS and so implementation is hindered by an inability to decide rationally what the best technology to use is. So, how can this powerful, data-rich technology be used to provide clinically relevant information for diagnosis and treatment? The diagnosis of infectious diseases is a process that begins with assessment of a patient by observation and interview together with nonspecific screening tests, for example urine dip-stick technology. This is often accompanied by the collection of specimens for processing in a microbiology laboratory where specific tests are performed to confirm the presence of markers of the immune response and/or the presence of microbial pathogens. It is in the generation of this specific

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Academic Clinical Fellow Microbiology, Norfolk and Norwich University Hospital, UK

has been successfully used in HIV and hepatitis B virus and is currently being implemented for other viruses along with barcoding and multiplexing to increase cost–effectiveness.”

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Author for correspondence: Department of Translational Microbiology, Norwich Medical School, University of East Anglia, Norfolk, UK Tel.: +44 016 035 67597 [email protected]

test data that NGS could, perhaps, find its place in the overall process of diagnosis. For viral infections, laboratory tests include the detection of antibodies by serology, which represents a significant workload, and the detection of viral pathogens, traditionally by electron microscopy and cell culture, and more recently by PCR [6] . Automation is common and the use of PCR, for some viral pathogens, directly on clinical specimens is incorporated into the majority of clinical laboratories. The impact of NGS in virology is increasing with several applications including in routine clinical work [6] . The preparation and purification of the nucleic acid, both DNA and RNA, is important and this may represent different challenges from different specimens. Normally, sterile sites, such as cerebrospinal fluid, are less likely to contain other genetic material, whereas samples from the gut will include large amounts of both human and microbial nucleic acids. For the sequencing of larger viral genomes, methods of prior virus enrichment are applied such as hybridization and PCR. Thus, for implementation of NGS as a diagnostic test, technical challenges remain but also the estimation of time/cost outcome needs to be carried out for each sample type and disease syndrome. In virology, this is underway; NGS has been used for sequencing full viral genomes for biodiversity for both large genomes (e.g., marseillevirus) and small genomes (e.g., influenza virus). In terms of monitoring resistance, NGS has been

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John Wain

Keywords : diagnostics • infectious disease • medical microbiology • next-generation sequencing • translational medicine

www.expert-reviews.com

10.1586/ERM.13.8

© 2013 Expert Reviews Ltd

ISSN 1473-7159

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Editorial

Wain & Mavrogiorgou

successfully used in HIV and hepatitis B virus and is currently being implemented for other viruses along with barcoding and multiplexing to increase cost–effectiveness. The rapid identification and control of outbreaks (e.g., for norovirus) is one area NGS carried out directly from clinical specimens could clearly aid public health and infection control agencies [6] .

“The use of next-generation sequencing … directly

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For bacterial and fungal infections laboratory diagnosis again involves the detection of immune response not only by serology but also by microscopy for immune cells while pathogen detection is via culture on agar plates and increasingly, for a minority of pathogens, by PCR; this includes screening tests for methicillinresistant Staphylococcus aureus and Clostridium difficile directly on clinical samples [7] . The use of mass spectrometry to identify bacterial cultures is being used increasingly and is driving the introduction of automation. In this context, the impact of NGS has so far been limited but is revolutionizing epidemiological analysis of cultures for infection control and outbreak detection for a limited number of pathogens. Several groups are using NGS to map outbreaks, most recently for Legionella infection [8] , salmonellosis [9] , methicillin-resistant S. aureus infection and carriage [10] and community acquired food-borne pathogens [11] . Perhaps the early wins for NGS in microbiology will be infection control, epidemiology in the community and targeted diagnostics [1] or the identification of isolated cultures [12] . However, while necessary for the stepwise progression toward the use of NGS for general diagnostics it is only the use of these powerful technologies directly in clinical specimens which will provide the timely information needed to affect clinical management of patients. The recognition of a bacterial cause of infection can guide clinicians towards broad spectrum antibiotics but these are fast running out and the use of more targeted therapies is clearly desirable. If NGS data can be used to identify not just the bacterial cause of an infection but also the genes responsible for resistance then antibiotic stewardship can also be improved and would represent a major step forward in clinical medicine. For parasitic infections, microscopy remains the technology used by the majority of front line laboratories but typing increasingly uses sequence-based methods. For a Cryptosporidium sample, sequencing is possible directly from water [13] and the use of direct NGS is being discussed [14] ; for malarial diagnosis, the depletion of human DNA greatly improves recovery of parasite

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in clinical specimens will provide the timely information needed to affect clinical management of patients.”

DNA directly from human blood [15] and transcription profiling methods have been developed [16] . It seems therefore that everything is present to solve the puzzle of direct detection of parasites (and other microbes) from clinical and environmental samples, we just need to assemble the separate components in an appropriate, cost effective, way. One major advantage of NGS is that it can be used to detect, and type, all organisms that contain nucleic acid from any specimen. It represents electron microscopy, tissue culture and the agar plate all rolled into one. Clearly for the implementation of NGS, as with any new diagnostic method, standardization is necessary [7] not least in the clinical interpretation of these data. Within microbiology, as in other medical fields such as oncology, complex results are explained to patients by clinical experts. Using NGS has the capacity to revolutionize microbiology but there will be major issues in the ethics as well as the science of the situation; NGS technology will result in incidental findings (past exposure or latent infections) and these, as in oncology [17] , will have to be dealt with. To maximize the potential for diagnosis, we will need clinical genomics, transcription profiling and microbial genomics to come together – these results, as with all clinical test results, will only provide a probability of a diagnosis, if the clinical picture does not match the result then the result should be questioned. As we move into the genomic era, we need to equip healthcare professionals with the knowledge to generate the confidence to use and interpret the results generated by the new technologies. Yes, we need to resolve the current technical barriers to the introduction of NGS but we also need to produce a new generation of scientists and clinicians who can interpret the very complex datasets generated by the new hardware and software solutions and deliver them to patients who will be concerned about the implications not just for them or their child but possibly for future generations.

References 1

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Köser CU, Ellington MJ, Cartwright EJ et al. Routine use of microbial whole genome sequencing in diagnostic and public health microbiology. PLoS Pathog. 8(8), e1002824 (2012).

“Next-generation sequencing … represents electron microscopy, tissue culture and the agar plate all rolled into one.” Perhaps, the greatest challenge for the introduction into clinical practice of rapid tests based on direct nucleic acid sequencing is one of education. Financial & competing interests disclosure

J Wain is CSO of a drug discovery company, Discuva Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Mutz KO, Heilkenbrinker A, Lönne M, Walter JG, Stahl F. Transcriptome analysis using next-generation sequencing. Curr. Opin. Biotechnol. 24(1), 22–30 (2013).

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Loman NJ, Constantinidou C, Chan JZ et al. High-throughput bacterial genome

sequencing: an embarrassment of choice, a world of opportunity. Nat. Rev. Microbiol. 10(9), 599–606 (2012). 4

Loman NJ, Misra RV, Dallman TJ et al. Performance comparison of benchtop

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Köser CU, Holden MT, Ellington MJ et al. Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. N. Engl. J. Med. 366(24), 2267–2275 (2012).

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fragment length polymorphism analysis, direct sequencing, and cloning. Appl. Environ. Microbiol. 77(12), 3998–4007 (2011). 14

Jex AR, Smith HV, Nolan MJ et al. Cryptic parasite revealed improved prospects for treatment and control of human cryptosporidiosis through advanced technologies. Adv. Parasitol. 77, 141–173 (2011).

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Oyola SO, Gu Y, Manske M et al. Efficient depletion of host DNA contamination in malaria clinical sequencing. J. Clin. Microbiol. (2012).

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Cantacessi C, Campbell BE, Jex AR et al. Bioinformatics meets parasitology. Parasite Immunol. 34(5), 265–275 (2012).

Ruecker NJ, Hoffman RM, Chalmers RM, Neumann NF. Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction

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Biesecker LG, Burke W, Kohane I, Plon SE, Zimmern R. Next-generation sequencing in the clinic: are we ready? Nat. Rev. Genet. 13(11), 818–824 (2012).

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Wain J, Keddy KH, Hendriksen RS, Rubino S. Using next-generation sequencing to tackle non-typhoidal Salmonella infections. J. Infect. Dev. Ctries. 7(1), 1–5 (2013).

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