FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS

Transcription

1 FUTURE PROSPECTS IN MOLECULAR INFECTIOUS DISEASES DIAGNOSIS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC WS2-5

2 Digital PCR New approach to nucleic acid detection and quantification Unlike real-time quantitative PCR, quantifies DNA in a sample without the need for a standard curve Provides precise absolute quantification of nucleic acids

3 Life Technologies QuantStudio 12K QuantStudio 3D RainDance RainDrop Digital PCR Bio-Rad QX100 ddpcr Fluidigm BioMark HD

4 Digital PCR vs qpcr Kuypers J, Jerome KR Applications of digital PCR for clinical microbiology. J Clin Microbiol 55:

5 Characteristics of dpcr vs. qpcr Advantage Absolute quantification, no standard curve Improved interlaboratory commutability Less affected by sample inhibitors Less affected by poor amplification efficiency More precise Better detection of low-copy number variant Disadvantage Limited reaction mixture volume Smaller dynamic range Molecular dropout Less accurate quantification of large amplicons Lower throughput Limited multiplexing exacerbated if assay requires internal control More expensive instrumentation and reagents Higher risk for contamination More complex to perform Kuypers J, Jerome KR Applications of digital PCR for clinical microbiology. J Clin Microbiol 55:

6 Applications of dpcr in Clinical Microbiology Characteristic Absolute quantification without a standard curve Less affected by poor efficiency of amplification Less affected by inhibitors in the sample Good sensitivity and excellent precision Application Quantification of pathogen load, especially targets with no available reference material Calibration of reference standards for qpcr Accurate quantification of targets with high sequence variability Accurate quantification of samples with inhibitors Precise quantification for monitoring low pathogen loads Detection of rare mutations and alleles Reliable determination of fold change measurements Detection of endonuclease-mediated gene editing Kuypers J, Jerome KR Applications of digital PCR for clinical microbiology. J Clin Microbiol 55:

7 Sequencing Technology Amplify specific sequences Perform sequencing and computer-assisted analysis, and query genetic database Traditional Sanger Sequencing (target-specific primers) Next Generation/Whole Genome Sequencing (NGS/WGS)

8 Whole Genome NGS Initial investment and running costs of NGS have decreased dramatically during last decade Single protocol can be used for all pathogens for both identification and typing No need for target-specific primers Whole genome of pathogen is sequenced at random following fragmentation of genome Exceptions Oxford Nanopore MinION and Pacific Biosciences Sequel can sequence in one piece Can be used to answer different diagnostic questions

9 Clinical Value of NGS: Agnostic Testing Has potential to dramatically revolutionize clinical infectious diseases testing Ultimate pathogen multiplex assay Identify any expected or unexpected pathogens from single specimen or as isolates Identify rare pathogens not frequently on differential Identify novel, highly divergent pathogens from a sample (metagenomics) Detect virulence determinants and genetic markers/variants of drug resistance Track outbreaks of infection

10 Next-Generation Desktop Sequencers NextSeg miseg Ion PGM miniseq Ion Proton Ion S5 MiniION

11 Properties of Current NGS Paltforms Company Equipment Output/run (Gb) Maximum read length (bp) Reads (x10 6 ) Running time Illumina MiniSeq x h Illumina Miseg x h Illumina NextSeq x / h Illumina HiSeq x <1-3.5 days ThermoFisher Ion PGM / h ThermoFisher Ion 5S h ThermoFisher Ion 5S XL <24 h Oxford Nanopore MinION , , min-48 h Pacific Biosciences* Sequel >20, , min-6 h Pacific Biosciences* RSII >20,000 55, min-4 h *Pacific Biosciences data are per smart cell; both Sequel and RSII can run 1-16 smart cells in one run Deurenberg RH et al Application of next generation sequencing in clinical microbiology and infection prevention. J Biotechnol 243:16-24

12 Ion Semiconductor Sequencing First post-light sequencer Ion 314 Ion 316 Ion 318 Powered by Scalable Silicon Chips Personal Genome Machine (PGM) Torrent Server and Databases Ion Torrent/Life Technologies

13 Simple Natural Chemistry

14 Ion PGM: Fast Direct Detection dntp H + Sensing Layer Sensor Plate ph Q V DNA Ions Sequence Nucleotides flow sequentially over Ion semiconductor chip One sensor per well per sequencing reaction Direct detection of natural DNA extension Millions of sequencing reactions per chip Fast cycle time, real time detection Directly translates chemical signals into digital information Bulk Drain Source Silicon Substrate To column receiver

15 Nanopore Sequencers 500 nanopores GridION Node 2,000 nanopores

16 Nanopore Sequencing Oxford Nanopore Technologies, U.K. Involves threading long, unbroken strands of DNA or RNA through a membrane pore using an enzyme, and measuring resultant changes in electrical current as nucleotide bases pass through it Nanpore chips can process DNA at a rate 0f bases/second/pore No sample amplification necessary

17 Nanopore Sequencing

18 Mainstreaming of Sequencing Routine clinical testing will be a reality with time Need for development of simplified solutions for all phases of testing Sample preparation Sequencing Data analysis Result interpretation Need to address clinical relevance of finding a fragment of nucleic acid that may not correlate with patient s disease Data analyses is biggest challenge; need access to well-vetted databases

19 Software Packages for NGS Analyses Deurenberg RH et al Application of next generation sequencing in clinical microbiology and infection prevention. J Biotechnol 243:16-24

20 MALDI-TOF MS in Clinical Microbiology Matrix-assisted laser desorption/ionization timeof-flight mass spectrometry Based on the detection of the mass of molecules Until now, main use has been to identify and type bacteria from a positive culture; used some for fungi, mycobacteria, and parasites Rapid and inexpensive; accurate results with simple sample preparation and minimal technical time Demonstrated applications in Clinical Virology

21 MALDI-TOF MS Clark EA, Kaleta EJ, Arora A, Wolk DM Clin Microbiol Rev 26:

22 MALDI-TOF MS Systems Bruker MALDI Biotyper CA System BioMerieux Vitek MS

23 MALDI-TOF MS Applications in Virology Identification of viruses from clinical samples Detection of mutations and strain variation Screening of viral subtypes Identification of antiviral resistance Epidemiology of viral infections

24 Host Response Diagnostics Analysis of transcriptome of host Transcriptome is full range of mrnas produced in a particular cell or tissue or expressed by an organism Can measure the expression of thousands of genes at the same time Generation of gene expression profiles can describe changes in the host transcriptome in response to a particular condition or treatment Can be coupled with metabolomics and/or proteomics

25 PLoS Genetics :e

26 Huang Y et al PLoS Genetics 7:e

27 PNAS : PLoS One :e52198 Nature Scientific Reports :33752

28 J Infect Dis : SciTransMed :322ra11 Open Forum Infectious Diseases DOI: /ofid/ofw007

29 Ribosome Profiling Innovative technique Uses deep sequencing to monitor gene expression at the level of translation rather than transcription Providing novel insights into the identities and amounts of proteins being produced in cells infected with viruses J Virol

30 Conclusions Significant advances have been made in field of clinical virology over the years Continuous introduction of newer technologies Will need to be adequately trained on these methods and well informed about the availability and utility of such tests for optimum integration into clinical care Continuous education and regular interactions between clinical virologists and healthcare providers will be vital