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 panel
Good time to use
single virus test
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 RP
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
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 RVPv1
Virus
% 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
Luminex xTAG RVPv1
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
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
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.