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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)
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
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?
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
Viral 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
Source: http://www.cdc.gov/flu/weekly/
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 RPVirus % 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 1 100
Parainfluenza virus 2 92.3
Parainfluenza virus 3 100
Respiratory syncytial virus A 86.4
Respiratory syncytial virus B 100
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)
Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533
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
Virus % Sensitivity
Adenovirus 100
Influenza A 100
Infuenza A H1/09 100
Influenza A H3 100
Influenza B 100
Human Metapneumovirus 100
Parainfluenza virus 1 100
Parainfluenza virus 2 100
Parainfluenza virus 3 100
Respiratory syncytial virus A 100
Respiratory syncytial virus B 100
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 RVPv1Virus % Sensitivity
Adenovirus 74.3
Influenza A 100
Infuenza A H1/09 100
Influenza A H3 92.9
Influenza B 95.5
Human Metapneumovirus 100
Parainfluenza virus 1 100
Parainfluenza virus 2 100
Parainfluenza virus 3 100
Respiratory syncytial virus A 86.4
Respiratory syncytial virus B 92.9
Rhinovirus/Enterovirus 93.0
Source: Popowitch, et al. 2013. J. Clin. Microbiol. 51(5): 1528-1533
Source: Luminex (http://www.xtagrvp.com/public/userfiles/MLD-019-KPI-001.pdf)
Luminex xTAG RVPv1
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 FastVirus % 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 N/A
Parainfluenza virus 3 N/A
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
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.