Kessler: Molecular Diagnostics of Viruses Relevant in the Immunocompromised Patient. Wien, November 2012

Transcription

1 MOLECULAR DIAGNOSTICS OF VIRUSES RELEVANT IN THE IMMUNOCOMPROMISED PATIENT VIRUSES RELEVANT IN THE IMMUNOCOMPROMISED PATIENT EBV HHV6 (HHV7) (HHV8) HSV1 HSV2 Parvovirus B19 Polyomavirus BK (Polyomavirus JC) VZV VIRUSES RELEVANT IN THE IMMUNOCOMPROMISED PATIENT EDTA whole blood (EDTA tube) EBV HHV6 HSV1 HSV2 Parvovirus B19 VZV Throat washing (or BAL; sterile screw cap collection vessel) Urine (sterile screw cap collection vessel) Polyomavirus BK 1

2 ANALYTICAL STEPS Past Present Nucleic acid extraction (sample preparation) Amplification (PCR) Detection (hybridization) Nucleic acid extraction (sample preparation) Amplification / detection (qpcr) However, time-consuming preparation of PCR mixes / addition of eluates Lysis of the nucleic acid-containing specimen Removal of substances which might inhibit subsequent amplification while protecting nucleic acids from degradation Concentration of nucleic acids into a small volume Classic (manual) protocols Time-consuming Labor-intensive Susceptible to contamination Commercially available kits on automated platforms Effective recovery of nucleic acids Risk of contamination and potential hazards caused by toxic reagents insignificant 2

3 MAGNETIC GLASS PARTICLE TECHNOLOGY PRINCIPLE Lysis, Stabilization, Deproteinization Capture Purification Elution DNA or RNA Pathogen MAGNETIC GLASS PARTICLE TECHNOLOGY PRINCIPLE Silica Eluate Matrix Input vol 1 (µl) Elution vol (µl) EDTA WB Throat washing (or BAL); urine CSF; samples from infants (preferably) input vol = loading vol = processing vol 3

4 Highly desirable features adjustable in a single run: Different matrices Extraction of DNA / RNA Addition of ICs Variable input volumes (beware of silica particle overload!) Variable elution volumes PCR MIX PREP / ELUATE ADDITION EDTA whole blood (Block 1) Adenovirus EBV HHV6 HSV1 HSV2 Parvovirus B19 VZV Throat washing (or BAL; Block 2) Adenovirus Urine (Block 3) Adenovirus Polyomavirus BK Eluate input vol: 5 µl (AnDiaTec), 10 µl (altona + Argene/bioMerieux); samples of blocks must be extracted twice (Argene incl. IC, AnDiaTec incl. IC + altona) PCR MIX PREP / ELUATE ADDITION User-friendly qpcr vessels (preferably transparent 96-well plates) Automation highly desirable (combined nucleic acid extraction & PCR mix prep / eluate addition) Different viral assays in a single run Preferably transparent 96-well plates Preparation of different PCR mixes (according to the specific order programmable like a clinical chemistry analyzer) Working in parallel to nucleic acid extraction Printable working lists Immediate addition of eluates to PCR mixes 4

5 AMPLIFICATION / DETECTION AMPLIFICATION / DETECTION Screening a panel of viruses in an immunocompromised patient Introduction of a set of qpcrs performed with one cycling program in parallel in a single run Flexible testing of clinical samples for the presence of different viruses Providing unaltered limits of detection/quantitation NOTE: Molecular assays based on multiplex real-time PCR may be significantly impaired by worsened limits of detection/quantitation TIME TO RESULT Morning ward round Sample collection Around Samples arrive in lab Afternoon ward round Sample collection Around Samples arrive in lab Time frame: 4 hrs 5

6 URGENT NEEDS Flexible and rapid extraction device Different matrices in a single run Variable input and elution volumes in a single run Automated and rapid preparation device for preparation of PCR mixes and addition of eluates (preferably transparent 96-well plates) Programmable for different parameters Working in parallel to nucleic acid extraction Open for different qpcr assays from different manufacturers Rapid qpcr instrument ISMD 2012 ISMD

7 ISMD 2012 MD LAB & RESEARCH UNIT MD Margit - Petra Brigitte - Harald THANK YOU FOR YOUR ATTENTION! Michael - Bettina - Evelyn 7