Molecular Diagnosis of Infectious Diseases Gregory J. Tsongalis, Ph.D.

Transcription

1 Molecular Diagnosis of Infectious Diseases Gregory J. Tsongalis, Ph.D. Professor of Pathology Director, Molecular Pathology Dartmouth Medical School Dartmouth Hitchcock Medical Center Norris Cotton Cancer Center Lebanon, NH

2 CANADA DHMC, Lebanon, NH * * California Boston, MA Texas Florida

3 The Human Genome Project February 2001 A major impact on microbial genomics.

4 Microbial Genomics 1995, first complete genome sequence of a free-living organism, Haemophilus influenzae Since then 1,554 complete bacterial genomes Species pangenome contains set of core genes that are common and set of dispensable genes that are absent in at least one strain. Approximately 90% of bacterial genome codes for protein whereas <2% of human genome codes for protein Relman DA. N Engl J Med 2011;365:

5 Why Molecular Pathology? Genetics previously unavailable tests carrier detection risk assessment Infectious diseases turn-around time inability to culture microscopic interpretation and competency quantitative analysis genotyping Heme/Oncology confirmation minimal residual disease therapeutics Identity testing most polymorphic molecule Therapeutics/PGx

6 Applications of Molecular Analyses to Infectious Diseases Qualitative detection Quantitative detection Identification/speciation Microbial Identity Testing Genotyping/Drug Resistance

7 Molecular Infectious Disease (Molecular Testing Methods) Nucleic acid (DNA, RNA) specimens: Blood CSF Tissue (fresh, frozen FFPE) Urine Stool Amniotic fluid Other body fluids Microbial isolate

8 Nucleic Acid Extraction Technologies Manual Extractions: 1. Make your own buffers 2. ph your own buffers 3. Lab math 4. TAT = 1.5 days 5. Low throughput Automated Extractions: 1. Reagent contracts 2. Consumables 3. Maintenance contracts 4. TAT = minutes 5. Higher throughput

9 Automation of NA Extraction Extracton Type Magnetic silica particles Magnetic charge switch particles Silica vacuum manifold plate Iron oxide particles Silica spin columns Instrument COBAS AmpliPrep, EZI, MagnaPure Compact and LC, Maxwell 16, NucliSens EasyMag iprep Purification Instrument 6100/6700 Automated Nucleic Acid Workstation, QIAxtractor m2000sp QIAcube Persing, et.al Molecular Microbiology Diagnostic Principles and Practice (2 nd Ed.) C. Hill, p123.

10 Extraction Issues to be Considered Specimen type Extraction Method Inhibitors Target sequences (DNA vs RNA) Controls (type and number)

11 Molecular Infectious Disease (Molecular Testing Methods) Nucleic acid probes for culture confirmation and direct detection (DNA and RNA probes) Nucleic acid amplification ( NAT ) technologies Sequence amplification Polymerase chain reaction (PCR) and real time PCR Transcription-mediated amplification (TMA and NASBA) Strand displacement amplification (SDA) Ligase chain reaction (LCR) Loop-Mediated Isothermal Amplification (LAMP) Signal amplification Branched-chain DNA (bdna) Hybrid capture Invader (Cleavase) Sequencing Sanger or Next Generation Microarrays and Mass Spectrometry

12 Controls for Qualitative Molecular Diagnostic Testing Sample Preparation Amplification Detection Extraction control (pre- or post-)? efficiency inhibitors Blank control Negative control Positive control Internal amplification control (IAC) (competitive vs non-competitive)

13 Real-Time PCR Permits rapid target Id; <30 min Eliminate post-pcr processing Highly specific; Hybridization probes Allows multiplexing Permits quantification Signal intensity is directly proportional to the amount of amplified DNA Threshold cycle (Ct) determination; the cycle at which target is first detected

14 Detection Chemistries Intercalating/binding dyes SYBR green I Dual label probes/ Quenched probes -TaqMan (5 nuclease assay) -Molecular beacons, Scorpions -FRET probes

15 Increase in Reporter Signal Reports Amplification of Target

16 Ct: Primary Signal Analysis Threshold Cycle Threshold Value Threshold penetration Threshold Line

17 Melt Curve Analysis Following amplification temperature is slowly increased (ie. 60 o c to 95 o c, 0.2 o c/sec) Strands denature Dye released, fluorescence decreases Melting curve; Temperature (x) vs. Fluorescence (y) Tm function of %GC and length

18 Melt Curve Analysis Target Phase Transition Primer dimer

19 Melt Curve Analysis JCV BKV

20 PCR Testing Steps Sample Preparation Amplification Detection/Analysis Automated extraction + Real Time PCR = STAT DNA Analysis

21 Molecular Infectious Disease (Qualitative) Positive or negative Increased sensitivity Decreased TAT Low to high throughput Becoming more automated Applications are limitless but questionable clinical utility Major impact with real time PCR

22 Qualitative Molecular Infectious Disease Testing (Chlamydia trachomatis) THE SILENT EPIDEMIC Misperception of prevalence Asymptomatic Non-specific symptoms Coinfections Unreliable diagnostic tests Asymptomatic individuals serve as a reservoir of infection Issues: high volume, STD (social), high throughput

23 Abbott LCx System Sample Prep Amplification Detection LCx Thermocycler LCx Analyzer

24 LCR Amplification (LCx MEIA System) MEIA particle linked to enzymatic conversion of fluorescent dyes

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26 BD Viper (ProbeTec System) GenProbe TIGRIS DTS

27 Molecular Infectious Disease Molecular Diagnostic Methods for C. trachomatis M2000 v2 (Abbott) ProbeTec (BD) Assay Gene Target NAAT Amp. Control Hybrid Capture II (Digene) PACE 2 CT (Gen-Probe) Plasmid (2 targets) Real-time PCR Yes Plasmid SDA Yes Plasmid and genome Hybridization No 23S rrna Hybridization No Aptima Combo 2 23S rrna TMA No Aptima CT 16S rrna TMA No TaqMan48 v2 Plasmid and omp1 PCR Yes

28 Qualitative Molecular Infectious Disease Testing (Non-Kit Based Assays) Home brew (Laboratory Developed Tests) Analyte Specific Reagents Primers > PCR > Gel Real time PCR Near patient testing???

29 Qualitative Molecular Infectious Disease Testing (EBV) Bam HI-W repetitive region (296 bp) Conserved Among EBV Strains

30 NEGATIVE POSITIVE SPECIMEN 1 SPECIMEN 1 SPECIMEN 2 SPECIMEN 2 DNA MARKER EBV BETA-ACTIN

31 Real Time PCR Detection of EBV Target gene = BNT p143orf38 EBV DNA, 5000, 500, 50 and 5 copies/ml

32 Molecular Infectious Disease (Bordetella pertussis) PCR is both sensitive and specific Viability (post treatment still detectable) Use of multiple primer sets Specimen types for PCR testing Nasopharyngeal swab (Dacron not Ca alginate or use of a mucolytic agent to avoid PCR inhibition) Other specimens may serve as alternatives Other laboratory testing (Culture or DFA) Bordet and Gengou (Starch Blood Agar) Antibiotic selection plate (Cephalexin) Synthetic media

33 Bordetella Target Genes Copy# pertussis parapertussis holmesii IS ,000 present not present present IS not present present present PTxs1 1 present present not present

34 Qualitative Molecular Infectious Disease Testing (What did we learn?) Specimen types Extraction efficiencies Automation vs manual Importance of controls Assessing performance characteristics Sensitivity, specificity, precision, accuracy Limit of blank, limit of detection Armbruster and Pry. Clin Biochem Rev 2008;29 (Si):S49- S52 Regulatory: FDA vs ASR vs RUO

35 Molecular Infectious Disease (Quantitative) How much is present Limit of quantification (LoQ) Automation Low to high throughput Which applications are clinically relevant Is the LLoQ the same as LoD? Why quant vs qual?

36 Molecular Infectious Disease (HIV-1) Qualitative Quantitative Genotyping

37 HIV-1 Subtypes Group M (Major) Subtypes A - H Group O (Outlier) Group N (New)

38 HIV-1 Viral Load Testing Not diagnostic (?) Designed to monitor infection Sensitivity may be higher than proviral DNA PCR Indications Disease progression Prognosis Response to antiviral agents 0.5 log 10 units is considered clinically significant

39 Molecular Assays for the Qualitative Detection of HIV-1 RNA Assay Method Target Application COBAS AmpliScreen HIV-1 Test, v1.5 (IVD) COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative Test (RUO) RT-PCR HIV-1 gag gene Qualitative detection of viral RNA from plasma, organs and tissues PCR HIV-1 gag gene Qualitative detection of HIV-1 RNA and proviral DNA in plasma, anticoagulated fresh whole blood and dried blood spots COBAS TaqScreen MPX Test (IVD) PCR Multiple Simultaneous testing for multiple viruses in a single sample: HIV-1 group M, HIV-1 group O, HIV-2, HCV and HBV

40 Molecular Assays for Quantification of HIV-1 RNA Assay Method Target Dynamic Range (copies/ml) Roche Amplicor HIV Monitor v1.5 test, Standard (IVD) Roche Amplicor HIV Monitor v1.5 test, Ultrasensitive (IVD) Roche Cobas AmpliPrep/Cobas TaqMan HIV Test v.1.0 (IVD) Roche Cobas AmpliPrep/Cobas TaqMan HIV Test v.2.0 (IVD) Siemens Versant HIV RNA 3.0 Assay (IVD) Biomerieux NucliSENS EasyQ HIV-1 V2.0 (RUO) Abbott RealTime HIV-1 Assay (IVD) RT-PCR HIV-1 gag gene ,000 RT-PCR HIV-1 gag gene ,000 Real-time PCR - TaqMan HIV-1 gag gene 48 10,000,000 Real-time PCR - TaqMan HIV-1 gag gene, LTR 20 10,000,000 bdna HIV-1 pol gene ,000 NASBA HIV-1 gag gene 25 10,000,000 Real-time PCR HIV-1 integrase gene 40 10,000,000

41 Viral Load Testing Interpretation of Results Results should be presented as Log 10 Values Prevents clinicians from over interpreting small variations in viral load Changes in viral load must exceed 0.5 log 10 (3 fold) to represent biologically relevant changes in viral replication Due to differences in performance, viral load should be quantified at follow-up by the same version of the same assay that was used initially

42 Viral Load Testing Interpretation of Results Clinical infections can increase viral load by as much as 1 log HSV infections Opportunistic infections Vaccinations Influenza Tetanus Pneumococcal Viral load testing should not be performed for 1 month following such infections

43 copies/ml) Patient Monitoring Initiate Rx Resistance Testing Viral Load (log Compliance 2. Resistance /99 2/99 3/99 5/99 8/99 12/99 4/00 5/00 9/00 2/02 6/02 7/02 8/02

44 HIV-1 Resistance Genotyping Determine the sequence RT and protease genes VGI (FDA approved), ABI, laboratory developed Compare sequence to wild type virus Identify mutations Associate mutations with resistance Knowledge of genetics of resistance Data base, rules based system Different interpretations of same mutations

45 Viral Genotyping Antiretroviral Resistance Testing FDA Approved Systems Assay Name Manufacturer Methodology Truegene HIV-1 Genotyping Kit ViroSeq HIV-1 Genotyping System Siemens Medical Solutions Diagnostics, Tarrytown, NY Abbott Molecular, Des Plaines, IL DNA Sequencing (polyacrylamide gel electrophoresis) DNA Sequencing (Capillary electrophoresis)

46 Viral Genotyping

47 The BK Virus BKV is a member of the polyomavirus family. BKV typically presents with respiratory infection and about 80% of adults are seropositive for BKV antibodies. After primary infection, the virus enters a latent phase in the kidneys, brain, and uterus. The virus usually remains dormant but may reactivate during pregancy, HIV infection, diabetes, or after transplant surgery.

48 BKV Reactivation with Immunosupression Following Bone Marrow Transplant Hemorrhagic Cystitis Following Renal Transplant polyomavirusassociation nephropathy (PVAN or BKVN) Rejected kidney transplant exhibiting BK nephropathy and chronic rejection.

49 Typical intranuclear inclusion pre- and post- micro-dissection a b

50 -df/dt -dt/dt Melt Curve Analysis for BKV/JCV From Microdissected Samples A B Glom Tubules 80 BK virus 60 JC virus 40 NTC Temperature Glom Tubules 80 BK Virus 60 JC Virus 40 NTC Temperature Adeyi OA, Belloni DR, Dufresne SD, Schned AR, Tsongalis GJ. Real-time polymerase chain reaction and laser capture microdissection techniques in the diagnosis of BK virus infection of renal allografts. Am J Clin Pathol 124(4): , 2005

51 Monitoring with PCR BK Viruria and Viremia relatively frequent in immunosuppressed renal transplants Positive PCR results alone not very useful Quantitative Testing (real-time PCR) BKV levels over time With viral level cut-offs BKV PCR useful for determining PVAN (Viscount 2007): In Urine: 100% Sensitivity; 78% Specificity In Plasma: 100% Sensitivity; 91% Specificity

52 Requirements for BKV PCR Quantitative Analytic Specificity: Only detects BKV Analytic Sensitivity: Low limit of detection? Wide dynamic range (concentrations above 10 log10 copies/ml) Specimen type: Urine and plasma

53 Plasma MGB vs. Eragen Assays

54 Urine MGB vs Eragen Assays

55 BKV PCR Obstacles No FDA-approved assays (IVDs): must use laboratory-developed tests (with ASRs) No international standards defining BKV concentrations Limited options for calibrators for standard curve-lack of standarization

56 BKV PCR Obstacles Urine specimens can be difficult: PCR inhibitors Assay should be specific for BKV but detect various strains of BKV equally (robust vs stringent) Gene target Primer/probe location

57 Quantitative Molecular Infectious Disease Testing (What did we learn?) Performance Variants Clinical implications QC of numerical data Automation

58 So what is next in molecular infectious disease testing?

59 Gram positive Bacillus (coccobacillus, diphtheroid ) Anaerobe Non-spore forming Slow grower Non-motile Diagnostic challenges of Propionibacterium acnes McPherson: Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed Saunders Elsevier Accessed Online, 8/31/11. Many strains are indole & catalase positive Frequent contaminant of blood cultures. Isabella Martin, M.D.

60 Habitat

61 Infections Three categories: Acnes in teenagers & adults. Invasive deep-seated infections Pacemakers, valves, shunts. Surgical wound infections Prosthetic joints, spinal hardware. Clinicaladvisor.com Accessed online 8/31/11. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 7th ed Churchill Livingstone Accessed online 8/31/2011.

62 Orthopaedics Quandary 1-2 years Ultra-sound guided joint aspiration with culture + - Implantation of new hardware 6 Wks IV Antibiotics Removal of hardware Revision Arthroplasty + - Oops! Good

63 PCR for P. acnes Real time PCR Taqman assay. Quantitative Ct detect contamination. Internal control: beta-actin? Multi-plexed?

64 The Automation Revolution Continues BD MAX System (HandyLab Jaguar) 1.fully automates cell lysis, nucleic acid extraction, PCR set-up, amplification and detection 2.24 samples per run Enigma Diagnostics Enigma ML 1. portable 2. self-contained 3. ultra-rapid, laboratorystandard results 4. point-of-care testing

65 Smaller is Better Idaho Technologies Film Array IQuum Liat Analyzer

66 PathoGenetix An automated system for identification and strain typing of pathogens in complex sample types. Does not require specific reagents for detection of each pathogen, this approach uses a single reagent set to create genomic barcodes which are then used to detect and identify thousands of strains from hundreds of species. No PCR or other amplification technique is applied. Genome Sequence Scanning (GSS) technology. GSS enables high throughput, single molecule DNA analysis to be used for pathogen identification and characterization in complex biological samples.

67 Biocartis Compact platforms whose ease of use will lower the entry barrier to diagnostic testing. Developing a platform that integrates sample preparation of nucleic acids, amplification, detection, and the generation of a result without user intervention and a detection platform that includes encoded micro carriers, a micofluidic cartridge, and an instrument for low to high multiplexing detection of biomarkers.

68 Lumora BART (Bioluminescent Assay in Real-Time) is a novel reporter system which is used with isothermal nucleic acid amplification technologies. Easy-to-use, affordable, robust hardware.

69 What if.. Miniaturize Low cost Fast Multiplex Smart phone ready

70 DHMC Molecular Pathology Laboratory and Translational Research Program Samantha Allen Betty Dokus Susan Gallagher Carol Hart Arnold Hawk Claudine Lefferts, Ph.D. Joel Lefferts, Ph.D. Rebecca O Meara Elizabeth Reader Mary Schwab Heather Steinmetz Laura Tafe, M.D. Brian Ward Eric York