Molecular Diagnosis Future Directions

Transcription

1 Molecular Diagnosis Future Directions Philip Cunningham NSW State Reference Laboratory for HIV/AIDS & Molecular Diagnostic Medicine Laboratory, SydPath St Vincent s Hospital Sydney Update on Molecular Diagnostics Detection of target genes of interest quantification Infectious diseases HIV Hepatitis C & B TB / MAC Cytomegalovirus Herpes simplex Varicella zoster CT/GC HPV Molecular Diagnostics Profiling mutations associated with disease outcome Hepatitis C genotype HIV drug resistance genotype Host genetic factors Thrombophilia CyP450 drug metabolism HLA type Technology Key developments Uptake in diagnostic arena Alternative methods other than PCR SDA, TMA, LCR, NASBA, bdna Availability of Analyte Specific Reagents (ASR) Real time or kinetic formats Automation Contamination control Key developments Key developments Diagnosis Monitoring Herpes simplex virus detection and differentiation Cytomegalovirus mrna or qdna Human papillomavirus (HPV) HCV / HIV primary infection Tx applications Bacterial sexually transmitted diseases Cytomegalovirus - response to therapy / relapse HIV drug resistance testing Hepatitis C RNA quantification and genotype Improved technology sensitivity / specificity / efficiency Personalized clinical management 1

2 Driving Product Development Source of error Pre-amplification Specimen integrity Nucleic acid extraction Reagent preparation Amplification Conserved target sequence primer design Equipment closed tube Contamination control Post-amplification Uncertainty Uncertainty of of Volumetric precision / accuracy End point detection gel, enzymatic, probe hybridisation Real time detection FRET, Taqman measurement measurement HIV RNA copies/ml Not detected AMPLICOR HIV-1 MONITOR Performance on Group M Subtypes Results on 20,000 Virus Particles/mL UG273 DJ258 DJ263 US1 US2 US3 US4 CM237 BK132 BZ167 ZB18 US278 P3100 SE364 SM145 SE365 UG270 UG274 CM235 CM238 CM240 CM243 POC30506 RA12 RA17 NP1465 BZ126 BZ162 BZ163 MIKA G A B C D Isolate and Subtype E F G Pre amplification MagNA Pure LC Isolation Principle - DNA Volumetric errors amplified Tedious manual, repetitive Specimen integrity Sample Addition of Lysis Buffer Addition of Addition of Magnetic Proteinase K Magnetic Separation Glass Particles Washing Magnetic Separation Elution Post amplification & detection Endpoint detection Volumetric error Time to result Calibration issues Result calculations 2

3 Monitoring in Real time Kinetic / real time product detection Agarose Gel Blotting FRET Real Time PCR Real Time analysis Starting viral load Low Yield Medium High Threshold Cycle number Real-time Detection in NASBA Target specific molecular beacons sense RNA oligo P1 RNase H oligo P1 Reverse Transcriptase RT RT RNase H & oligo P2 Reverse Transcriptase oligo P2 anti-sense RNA T7 RNAP T7 RNA polymerase Molecular beacon hybridization 3

4 NASBA Calibrators & Quantitation Real Time Target Amplification NucliSens HIV-1 QT NucliSens EasyQ HIV-1 Log signal Qa Qb Qc Fluorescence WT Qd Log copies End-point measurement (WT and 3 calibrators) Time Kinetic measurement (WT and 1 calibrator) Mutation Detection Probe Design perfect match Mutation detection and product analysis mismatch Anchor Probe Mutation Probe T m of Mutation Probe approx. 5 C lower than T m of Anchor Probe Variation in Product T m Melting Curve product analysis T m varies by GC composition Low GC High GC temperature T m is influenced by: Salt concentration MgCl 2 concentration SYBR Green I concentration 4

5 Herpes simplex type differentiation IC detection Internal control inhibitor control Breakthrough of resistance HIV drug resistance testing 5

6 Reverse hybridisation Linear Array Assays 6

7 Line probe hybridisation HCV genotype Genotype 1 2 Subtype 1a 1b 2a Prevalence approx55% isolates Geography USA, Europe USA, Europe, Japan (73%) ; South Korea; China; Taiwan Nth USA, Italian and Japanese immigrants Response to therapy Difficult Most difficult a-ifn 2b Nth USA, Italian and Japanese immigrants Favourable (2-3 x higher than 1) 2c Italy 3 3a 3b approx 35% isolates IVDU USA, Europe, Indian subcontinent; Australian; Thailand; Myanmar; Nepal Favourable (2-3 x higher than 1) 4 Nth Africa; Middle East; Egypt Difficult, resemble 1 5 South Africa Difficult, resemble 1 6 Hong Kong Vietnam; Thailand; Indonesia Difficult, resemble 1 7 Vietnam; (Thailand; Indonesia) 8 Vietnam; (Thailand; Indonesia) 9 Vietnam; (Thailand; Indonesia) 10 10a Indonesia 11 Indonesia Kinetics of HIV-therapy Individualised management of HIV infection Baseline Efficacy pre-existing mutations A B Turnover High prevalence Full resistant Full replication * B C Low prevalence Full resistant Full replication Low prevalence Replication compromised 100% inhibition replication incompetent prevalence zero * Additional mutations -Resistance -Replication Algorithm for assessment of antiviral therapy Personalized Medicine HIV infection Combination therapy Initial response >1 log in HIV-1 RNA < 1 log in > 1-2 log in HIV-1 RNA HIV-1 RNA Yes Poor compliance? No Patient education Lack of drug efficacy? (due to patient s sub-optimum pharmacokinetic parameters) Continue therapy Yes No Modify/change therapy Predisposition to illness: (genetic testing) Diagnose and intervene before illness occurs: HIV serology, viral load and CD4 cell count Disease Prognosis/Therapy Selection: HIV viral load, CD4 cell count, and drug resistance testing (proviral DNA, genetic testing) Medical Intervention: HAART Enhanced monitoring: HIV viral load, resistance testing (proviral DNA) Resistant virus Assay for drug resistance Healthy Years Latent Disease Active Disease Chronic Disease Change drug combination Change therapy Continue therapy HIV infection Highly Active Antiretroviral Therapy 7

8 Personalized Medicine HCV Infection Real time HCV assay Individualised management of HCV infection HCV Qual HCV Quant Genotype confirm Quantity & Strain viremia of Virus 10 6 viral load HCV Quant 12 week response Pegylated IFN treatment Stop? HCV Qual response at end of treatment HCV Qual response at end of follow-up Relapse HCV Qual prolonged follow-up HCV-RNA levels SVR undetectable Wk 0 HCV genotype 1 (or 4) 48 wks 72 wks years 8