Rhinovirus CPE in fibroblasts

Transcription

1 The Molecular Evolution of Clinical Virology The University of Michigan Story No conflicts of interest to disclose Duane W. Newton, Ph.D., D(ABMM) Associate Professor of Pathology Director, Clinical Microbiology Laboratories Associate Director, Division of Clinical Pathology Diagnostic virology Direct detection EIA DFA Molecular Culture Tube Shell vial Serology UM Clinical Virology DFA + Tube Culture + Serology DFA + Tube Culture + Shell Vial + Serology DFA + Shell Vial + Molecular Molecular Direct Immunofluorescence Assays (DFA) NP swabs Respiratory viruses Flu, RSV, Paraflu, Adeno Lesions HSV and VZV Viral Culture Tube cultures 1 tube MRC-5 (fibroblast line) 1 tube A549 (carcinoma line) 2 tubes Monkey Kidney cells (primary cells) 1 tube Rabbit Kidney cells (added for HSV) 1 tube HEp-2 (added for RSV) 1 tube of BGM and/or RD (added for EV) TAT 2-4 hrs, day shift M-F non-winter Sat and Sun during winter microgenbioproducts.com Incubation: 2-4 weeks at 34 or 37ºC Hemadsorption of RMK at different times (days 3 & 7) CPE+ type by FA (most of the time) HAD+ type by FA 1

2 Rhinovirus CPE in fibroblasts Courtesy of Dan Wiedbrauk, ASM image library Shell vial cultures Viral culture volumes CMV: 2 MRC-5, 37ºC, 24 & 48 hr VZV: 2 MRC-5, 37ºC, 48 & 96 hr Adeno: 2 MRC-5, 37ºC, 48 & 72 hr HSV: 2 MRC-5, 37ºC, 24 & 48 hr 2

3 Why use molecular methods? When conventional methods are: Too cumbersome or slow (e.g., virus isolation) Too insensitive (asymptomatic HIV, viral infection of central nervous system, etc.) Not available (unculturable agents: HPV, HCV) When did we make changes? Clinical need New platforms Expansion of menu Change in performance 3

4 HSV non-genital Lesions HSV CSF Focus Direct Future state Viral load assays HIV, HCV, CMV, HBV Roche COBAS Amplicor Roche AmpliPrep/TaqMan Abbott m2000 sp/rt Qualitative assays CMV non-blood Viral load assays EBV BK virus Adenovirus Future state 4

5 UM Virology, ca Traditional tube culture for respiratory viruses Replaced with shell vials in 2008 Decreased detection time from up to 14 days to hr. Influenza A, B, RSV, parainfluenza 1,2,3, adenovirus, hmpv DFA for influenza A,B, RSV Planning to incorporate Flu/RSV PCR for season Pandemic Influenza, H1N1 was first detected in the United States in April 2009 unique combination of influenza virus genes never previously identified in either animals or humans April 26 August 2, specimens submitted for culture / DFA 226 influenza A UMHS Needs Assessment Test Development Lab challenged to develop a high volume molecular assay by October 1, 2009 Influenza A/B/RSV adapted to automated realtime platform LOD 10 viral particles Subtyping assay Pandemic H1N1 Seasonal H1N1 Seasonal H3N2 Solution Prodesse ProFlu+ assay on m2000 Further Advances Real time assays developed and implemented for: parainfluenza virus types 1, 2, 3 Prodesse ParaFlu+ human metapneumovirus Prodesse hmpv+ UMHS Options / Challenges Options PCRFL : influenza A/B PCRRSV: respiratory syncytial virus PCRSC: influenza A/B/RSV PCRIR: influenza A/B/RSV, parainfluenza 1, 2, 3, human metapneumovirus adenovirus culture by request Challenges Assay time to completion is 6.5 hours 70-80% of orders are for PCRIR 80-85% of positives are influenza A, B, or RSV rhinovirus, coronavirus, and adenovirus not detected Reassessment / New algorithm? Influenza A/B/RSV screen Focus Direct real-time PCR assay 55 minutes assay time LOD TCID50 seasonal H1 0.1 TCID H1N TCID50 influenza B 3.0 TCID50 RSV 5

6 BioFire Film Array Respiratory Panel BioFire multiplex array Direct real-time PCR assay 1 hour assay time adenovirus coronavirus HKU1 coronavirus NL63 coronavirus 229E coronavirus OC43 human metapneumovirus human rhinovirus/ enterovirus influenza A influenza A/H1 influenza A/H influenza A/H3 influenza B parainfluenza 1 parainfluenza 2 parainfluenza 3 parainfluenza 4 respiratory syncytial virus Bordetella pertussis Chlamydophila pneumoniae Mycoplasma pneumoniae Improved current state Performance improvements Adenovirus; expanded panel Turn-around time improvements Cost savings Reagent costs and reduced error rates Educational efforts required Emphasis on Flu A/B, RSV Full panel for inpatients only Future state BioFire FilmArray panels GI Positive blood cultures Cepheid GeneXpert Enterovirus (CSF) GC confirmation Mtb/rifampin resistance Staffing changes Molecular in micro, virology separate Overlap Molecular separate Molecular on multiple shifts 6

7 Space utilization UH Micro Mott UH and Mott More unidirectional flow Future state New space at NCRC 2 miles away Micro/Viro/Molecular together? Molecular ID with Molecular path? Role for culture? Persistent PCR positivity Influenza virus Emerging viruses EV68 7

8 Evolution of UM molecular In-house PCR HSV, meca, Pneumocystis Digene HC2 COBAS Amplicor CT/NG, HIV, HCV, HBV TECAN, MWP CT/NG LightCycler HSV AmpliPrep/TaqMan HIV, HCV, HBV BD Viper CT/NG Abbott m2000 respiratory viruses, HIV, HCV, HBV COBAS 4800 HPV BioFire respiratory pathogens Focus Flu/RSV, B. pertussis, C. difficile GeneXpert Enterovirus Thank you! 8