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HIV Drug Resistance Training

VIEWS: 1 PAGES: 27

									HIV Drug Resistance Training




         Module 5:
         Sequencing Procedures:
         Commercial and In-House

                                   1
                      Topics

   Overview of Genotyping Procedures
   Comparison of Three Different Procedures
   Application of Concepts




                                               2
                    Objectives

   Identify individual lab needs for sequencing.
   Identify differences among the procedures.
   Based on the differences among the procedures,
    identify the procedure that best meets the lab’s
    needs.




                                                       3
overview of genotyping
procedures
What systems are available for HIV DR genotyping?
What do these systems require?




                                                    4
        HIV DR Genotyping Systems

   US FDA-approved HIV-1 genotyping systems:
    – ViroSeq™ HIV-1 genotyping system
    – TRUGENE® HIV-1 genotyping system
   In-house, or “home-brew” genotyping systems




                                                  5
Which Procedures do You Currently Use?


                        ViroSeq   Trugene   In-house

 Viroseq only                
 Trugene only                         
 In-house only                                   
 ViroSeq and Trugene                 
 Viroseq and In-House                           
 Trugene and In-house                           
 All three                                     


                                                       6
            US FDA-approved Systems

   Characteristics:
    – Approved for HIV-1 subtype B viral DR genotyping *
    – Labor-intensive and very expensive
   Requirements:
    –   Specific instruments
    –   Specimen: EDTA plasma
    –   Viral load level: ≥1000-2000 copies/ml of plasma
    –   Extensive training for technicians
    –   Laboratory Biosafety level II working environment for
        sample preparation
     * They have been demonstrated to work well for non-B subtypes
     as well, although with less certainty around performance
     characteristics.                                                7
comparison of three different
procedures
What are the procedures used by ViroSeq™, TruGene®, and in-
house assays?
What factors should be considered in choosing one procedure over
another?




                                                                   8
Option One: ViroSeq™ HIV-1 Genotyping

   Detects HIV-1 subtype B viral resistance
    – in plasma samples
    – collected in EDTA tubes
    – with a viral load ranging from 2,000 to >750,000
      copies/ml.
   Genotypes the entire HIV-1 protease coding
    region (codons 1-99) and codons 1-335 of
    reverse transcriptase.




                                                         9
  ViroSeq™ HIV-1: Genotyping Procedure

                     RNA                    Reverse                                          Purification                  Cycle                      Automated




                                                                                                              Sequencing
Sample Preparation




                                   RT/PCR




                                                             Purification & Quantification




                                                                                                                                          Analysis
                     extraction             transcription                                    and                           Sequencing                sequence
                     from                   (RT) and                                         quantification                using an ABI              detection
                     plasma in              Polymerase                                       of RT-PCR                     9700                      using an ABI
                     BSL II hood            chain reaction                                   products                      thermocycler              3100 DNA
                                            (PCR) to                                                                                                 genetic
                                            amplify target                                                                                           analyzer
                                            gene region
                                            using an ABI
                                            9700
                                            thermocycler




                                                                                                                                                                    10
ViroSeq™ HIV-1: Genotyping Procedure




           Sequence                        Confirmation of              Phylogenetic              Generate drug            Review drug




                                                                                         Report
Analysis




                                                             Analysis




                                                                                                                  Review
                            Confirmation


           analysis using                  sequencing                   analysis to               resistance               resistance
           ViroSeq HIV-1                   analysis by 2nd              detect cross-             report                   report and
           genotyping                      technician                   contamination.                                     send the report
           system                                                                                                          back to
           software v2.7.                                                                                                  requester




                                                                                                                                        11
Option Two: TRUGENE® HIV-1 Genotyping

    Semi-automated technique
    Covers PR (codons 4-99) and RT (codons 38-248)
    Viral RNA extracted from plasma
     – Plasma viral load ≥1,000 copies/ml
    cDNA amplified in a single-tube RT-PCR reaction
    Sequenced with a modified Sanger technique
     – Dye-primer (not dye-labeled ddNTPs)
     – PR and RT sequences non-contiguous
  Semi-automated data analysis, alignment and
   interpretation
  Specific chemical waste disposal required
   (acrylamide)                                        12
TRUGENE® HIV-1: Genotyping Procedure



              Sample                      RT-PCR                       CLIP™                            Detection using




                                                          Sequencing
Preparation




                                 RT-PCR




                                                                                      Electrophoresis
              preparation:                transcription                Sequencing                       Long-Read
              Viral RNA                   and                          using an ABI                     Towers
              extraction using            amplification                9700                             • Cast acrylamide
              QIAamp viral                using an ABI                 thermocycler                       gels
              RNA Mini Kit or             9700                                                          • Prepare MicroCel™
                                                                                                          cassette and
              other                       thermocycler                                                    loading gel onto
              commercial kits                                                                             Long-Reader
                                                                                                          Tower




                                                                                                                          13
TRUGENE® HIV-1: Genotyping Procedure



                      Data                           Cross-                    Generate              Review drug




                                                                     Results
Sequence Evaluation




                                     Data Analysis




                                                                                            Review
                      verification                   contamination             genotyping            resistance
                                                     detection                 results               report and
                                                     using Genetic                                   send report
                                                     Fingerprint                                     back to
                                                     Confirmation                                    requester
                                                     of data
                                                     analysis by
                                                     2nd person




                                                                                                                   14
Home-brew/In-house Genotyping Assays

   Many home-brew genotyping assays
   Differences:
    – Detection of HIV-1 viral resistance for non-B subtypes
      and circulating recombinant forms (CRFs)
    – Specimen type: plasma, dried blood spots (DBS), or
      dried plasma/serum spots (DPS/DSS)
    – Boundaries of the protease and the reverse
      transcriptase regions of the pol gene that are
      sequenced
    – Sensitivity (viral load requirement)
    – Other performance characteristics (validation)
    – Cost and turn-around time
                                                               15
Example: Home-brew/In-house
    Genotyping Procedure




                              16
Example: Home-brew/In-house
    Genotyping Procedure




                              17
           Comparative Analysis : 1 of 4

                                                        CDC Home-
                     ViroSeqTM        TRUGENE®
                                                        brew assay
   US FDA
                        Yes               Yes                 No
  approved
                                                        Major group M
               FDA approved for    FDA approved for      viruses, A, B,
HIV-1 subtype subtype B, but may subtype B, but may      C, D F and G
              also work for non-B also work for non-B   and CRF01-AE
                                                        and CRF02-AG
Diversification
                  Semi-open system   Closed system      Open system
  possibility
Cost for major                                            $200,000-
                     $200,000           $90,000
 instruments                                              $275,000

                                                                      18
          Comparative Analysis : 2 of 4

                                                           CDC Home-
                      ViroSeqTM          TRUGENE®
                                                           brew assay
                     UNG system
     Cross                              Built in Genetic
                     (destroy PCR
 contamination                           Fingerprint for     varies
                      amplicons
    control                            sequence analysis
                   containing dUTP)
                                                           Moderately
 Labor intensity    Moderately high          High
                                                             high
                                                             Lots of
 Sequence data
                   Experience needed    Semi-automatic     experience
    analysis
                                                            needed
  Instrument
                       expensive          moderate          expensive
Maintenance cost

                                                                        19
           Comparative Analysis : 3 of 4

                                                            CDC Home-
                    ViroSeqTM           TRUGENE®
                                                            brew assay
                                                             Non-system
 Sequence data
                  System specific      System specific         specific
analysis software
                                                              software
                                     BSL II cabinet and
    Biosafety
                   BSL II cabinet     Specific chemical     BSL II cabinet
  requirement
                                    disposal requirement
                                        High including
Biohazard waste                      specific requirement
                     Moderate                                 Moderate
   generated                        for handling chemical
                                             waste
  Reliability of
                       Varies              Varies               Varies
    supplies
                                                                         20
          Comparative Analysis : 4 of 4

                                                           CDC Home-
                      ViroSeqTM           TRUGENE®
                                                           brew assay
Labor intensity                       Heavy, individual
                                                        Moderate, batch
  (sequence       Moderate, batch run patient sequence
                                                             run
  detection)                                 run
    Overall
                       Complex             Complex         Very complex
  complexity
                                                          Surveillance and
                   Surveillance and     Small to medium   medium to large
Cost-effective
                   medium to large          patient         population-
consideration
                   population-based       genotyping           based
   for use
                  genotyping services      services         genotyping
                                                              services

                                                                        21
                   Cost Comparison

                                         In House
                    ViroSeq   TRUGENE®
                                          (CDC)
    Cost/kit         $5688     $4292       N/A

 No. tests/kit        48         30        N/A

   Cost/test         $120       $150     $80-150

Actual Cost/test
(including 15%       $140       $165     $95-160
    repeats)




                                                    22
Summary of HIV-1 Genotyping Procedures


  Two US FDA-approved genotyping systems
  Many home-brew genotyping systems
  All are conventional viral population-based
   sequencing assays with a minor viral population
   detection limit of ≥20%
  Complex assays - need highly skilled staff,
   dedicated instrumentation and infrastructure
  Test and instrument maintenance are expensive



                                                     23
application of concepts

Which type of procedure is most appropriate for our national lab?




                                                                    24
                  Discussion

 What factors are most important in choosing a
  procedure?
 Which procedure (if any) meets your needs best?
 What issues do you need to resolve in order to
  obtain and use the procedure you have selected?




                                                    25
                    Reflection

   Which procedure will we use (if any)?
   What must we do as “next steps?”




                                            26
                    Summary

   Overview of Genotyping Procedures
   Comparison of Three Different Procedures
   Application of Concepts




                                               27

								
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