About these slides SPEC – Short Presentation in Emerging Concepts Provided by the CAP as an aid to pathologists to facilitate discussion on the topic Content has been reviewed by experts at the CAP, but does not necessarily reflect the official opinion of the College of American Pathologists. Non-CAP material with identified copyright source may only be copied or distributed under a license (permission) from the copyright holder, or under the doctrine of fair use. Version 1.0, rev. 12/31/2013

Emerging Concepts in the Diagnosis of Respiratory Viruses Short Presentation on Emerging Concept (SPEC)

Respiratory Viral Infections Respiratory infections account for ~4 million deaths per year, about half of which are due to viruses Common viruses can cause serious respiratory infections New viruses are also being identified Metapneumovirus (MPV) Severe acute respiratory syndrome coronavirus (SARS-CoV) Avian influenza viruses H5N1, H7N9 Coronaviruses NL63 and HKU1 Human bocavirus Middle East respiratory syndrome coronavirus (MERS-CoV) -Affect many patient populations, inpatient and outpatient -Young children, elderly, immunocompromised, and adults with underlying medical conditions are at greatest risk for serious disease -Multiple common viruses and bacteria cause serious respiratory infections (Influenza viruses A & B; Respiratory syncytial virus--Most frequent cause of hospitalization in children <5; Parainfluenza viruses 1, 2, & 3; Adenoviruses -A good source for more information: The Global Burden of Disease 2004 update, WHO: http://www.who.int/healthinfo/global_burden_disease/GBD_report_2004update_full.pdf

Why Identify the Virus? Many viruses have similar initial symptoms Some patients will quickly deteriorate, while others could be sent home to recuperate with reassurance Different viruses may require different isolation practices; allows hospital to utilize infection control practices where patients are separated into wards by virus type Important to distinguish viral from bacterial causes Avoid unnecessary antibiotics Select specific antiviral agents, if available By utilizing epidemiologic data from lab, can prescribe appropriate prophylactic treatments (influenza and RSV) when necessary for at risk patients

Example of laboratory tracking of viruses, helpful to know what is in “season.” Can generate this type of data from multiple single-virus assays or from a larger panel. Source: Kiechle, et al. Clin Chim Acta. 2013 May 31. pii: S0009-8981(13)00238-6.

Why Identify the Virus? As new pathogens emerge, the ability to exclude known viruses may help to more rapidly recognize and identify the presence of a new pathogen Possible cost savings: Shorter ER times for diagnosis/triage Quicker access to treatment Shorter hospital stays Ability to “cohort” patients to prevent sick patient from catching a second virus

Traditional Identification of Viral Pathogens Direct fluorescent-antibody assay and culture Time consuming (slow turn-around-time) Labor intensive/require expertise to interpret Require monoclonal antibodies for viruses (for rapid cell culture) Virus must be viable Direct antigen testing Quick results Sensitivity and specificity vary widely, usually less sensitive than culture Some are simple to use point-of-care tests

Molecular-Based Viral Identification PCR (DNA/RNA)-based assays are gaining popularity Quicker turn-around-time Increased sensitivity Quick development for emerging pathogens (does not rely on development of monoclonal antibody) Ability to multiplex

Respiratory Virus Panels Can multiplex relatively easily, with minimal increase in cost More readily identify co-infections Identify virus more quickly than ordering tests sequentially, particularly when there isn’t a prevalent virus “in season” Sometimes a new virus may “cross-react” with an existing panel virus, aiding in identification until a specific test is available Ability to exclude many viruses simultaneously

When should a viral panel be used vs. a single virus test? During epidemic when there is one (or few) major virus(es) circulating When a new/prevalent pathogen suspected is not on a panel, but has a specific test When demand for test is too high for throughput available with panel When there isn’t a single prevalent virus Follow CDC data In hospital setting when infection control measures must be implemented To rule out many viruses at once when a new virus is suspected

Good time to use single virus test Good time to use panel CDC has similar surveillance for other viruses as well Source: http://www.cdc.gov/flu/weekly/

Note to Pathologist Presenting The next slides will show specifics of several FDA-cleared viral panels Please note that although information about specific commercially available panels is contained in this presentation that this is for informational purposes only. This does not imply endorsement by CAP of any specific vendor panel. Select the slides relevant for the assays you offer in your laboratory and delete the remainder or insert a slide or two describing your laboratory-developed test and its specificity/sensitivity characteristics

Biofire FilmArray RP FDA-cleared Detection Methodology: Melting Curve Analysis Viruses Reported: Adenovirus; Coronavirus HKU1, NL63; Influenza A (H1/2009, H1, H3); Influenza B; Human metapneumovirus; Parainfluenza virus 1, 2, 3, 4; RSV; Rhinovirus/enterovirus Overall Sensitivity: 89.4% Overall Specificity: 99.6% Hands-on Time: 0.05 hour Time to Result: 1.2 hours # of samples per instrument in 8 hrs: 7 Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Biofire FilmArray RP Pros: Cons: Virus % Sensitivity Adenovirus 57.1 Influenza A 86.2 Infuenza A H1/09 73.3 Influenza A H3 100 Influenza B 77.3 Human Metapneumovirus 96.2 Parainfluenza virus 1 Parainfluenza virus 2 92.3 Parainfluenza virus 3 Respiratory syncytial virus A 86.4 Respiratory syncytial virus B Rhinovirus/Enterovirus 83.7 Pros: Quick turn-around time Great specificity (~100% for all targets) Extended FDA-cleared panel including several bacteria Chlamydophila pneumoniae Mycoplasma pneumoniae Bordetella pertussis Cons: Limited capacity (1 sample at a time) Sensitivity data from a recent study Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Data from the manufacturer Source: Biofire (http://www.biofiredx.com/pdfs/FilmArray/InfoSheet,%20FilmArray%20Respiratory%20Panel-0229.pdf)

Genmark eSensor RVP FDA-cleared Detection Method: Voltammetry Viruses Reported: Adenovirus (C, B/E); Influenza A (H1/2009, H1, H3); Influenza B; Human Metapneumovirus; Parainfluenza viruses 1, 2, 3; RSV (A, B); Human Rhinovirus Overall Sensitivity: 95.4% Overall Specificity: 99.7% Hands-on Time: 0.92 hour Time to Result: 7.2 hours # of Samples per Instrument in 8 hours: 21 Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Genmark eSensor RVP Pros: Cons: Very sensitive Virus % Sensitivity Adenovirus 100 Influenza A Infuenza A H1/09 Influenza A H3 Influenza B Human Metapneumovirus Parainfluenza virus 1 Parainfluenza virus 2 Parainfluenza virus 3 Respiratory syncytial virus A Respiratory syncytial virus B Rhinovirus 90.7 Pros: Very sensitive Can report adenovirus species (C vs. B/E) Cons: Does not detect enterovirus Time to result >7 hours Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Luminex xTAG RVPv1 FDA-cleared Detection Methodology: Fluroescence-labeled Bead Array Viruses Detected: Adenovirus; Influenza A (H1, H3); Influenza B; Human Metapneumovirus; Parainfluenza virus 1, 2, 3; RSV (A/B); Rhinovirus/enterovirus Sensitivity: 91.2% Specificity: 99.7% Hands-on time: 1.2 hours Time to Result: 7.8 hours # of Samples on Instrument in 8 hours: 21 Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Luminex xTAG RVPv1 Virus % Sensitivity Adenovirus 74.3 Influenza A 100 Infuenza A H1/09 Influenza A H3 92.9 Influenza B 95.5 Human Metapneumovirus Parainfluenza virus 1 Parainfluenza virus 2 Parainfluenza virus 3 Respiratory syncytial virus A 86.4 Respiratory syncytial virus B Rhinovirus/Enterovirus 93.0 Data from a recent study Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Luminex xTAG RVPv1 Senstivity data from the manufacturer Source: Luminex (http://www.xtagrvp.com/public/userfiles/MLD-019-KPI-001.pdf)

Luminex xTAG RVP Fast FDA-cleared Detection Methodology: Fluroescence-labeled Bead Array Viruses Detected: Adenovirus; Influenza A (H1, H3); Influenza B; Human metapneumovirus; RSV; Rhinovirus/enterovirus Sensitivity: 78.8% Specificity: 99.6% Hands-on Time: 0.75 hour Time to Result: 4.8 hours # of Samples per Instrument in 8 hours: 21 Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Luminex xTAG RVP Fast Pros Cons Quick time-to-result High throughput Virus % Sensitivity Adenovirus 82.9 Influenza A 86.7 Infuenza A H1/09 81.3 Influenza A H3 78.6 Influenza B 45.5 Human Metapneumovirus 100 Parainfluenza virus 1 N/A Parainfluenza virus 2 Parainfluenza virus 3 Respiratory syncytial virus A 86.4 Respiratory syncytial virus B 85.7 Rhinovirus/Enterovirus 93.0 Pros Quick time-to-result High throughput Cons No parainfluenza coverage Lower sensitivity Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533

Respiratory Panel Considerations Negative results do not exclude the possibility of infection with a respiratory virus as the virus could be below the assay limit of detection Positive results do not exclude the possibility of co-infection with other viruses or bacteria, or concurrent underlying pulmonary pathology

Respiratory Panel Considerations Specificity and sensitivity for each virus, throughput, and turn-around-time vary greatly among commercially available panels Unique characteristics of the patient population being treated must be considered in selecting a panel What viruses are my patients at risk for contracting? How timely does the result need to be received to clinically impact patient care? When multiple testing options are available, good communication between the laboratory and treating physicians is essential for optimal patient care Pathologist: This is a good opportunity to engage your clinicians in helping select a panel for your laboratory (if you don’t already have one in place) or a lab/panel for send-out tests. By understanding your local patient needs, you can provide better service for the clinicians and patients.

Selected Resources Popowitch, et al. Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP Fast Multiplex Assays for Detection of Respiratory Viruses. J. Clin. Microbiol. 2013, 51(5): 1528. Mahony, et al. Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay. J. Clin. Microbiol. 2007, 45(9): 2965. Griswold. Sizing up ‘mega’ multiplex panels for respiratory viruses. Cap Today. May 1, 2013.