1. 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

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

3. 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:

4. 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

5. 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 May 31. pii: S (13)

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

7. 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

8. 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

9. 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

10. 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

11. Good time to use single virus test
Good time to use panel
CDC has similar surveillance for other viruses as well
Source:

12. 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

13. 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 J. Clin. Microbiol. 51(5):

14. 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 J. Clin. Microbiol. 51(5):

15. Data from the manufacturer
Source: Biofire (

16. 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 J. Clin. Microbiol. 51(5):

17. 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 J. Clin. Microbiol. 51(5):

18. 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 J. Clin. Microbiol. 51(5):

19. 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 J. Clin. Microbiol. 51(5):

20. Luminex xTAG RVPv1
Senstivity data from the manufacturer
Source: Luminex (

21. 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 J. Clin. Microbiol. 51(5):

22. 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 J. Clin. Microbiol. 51(5):

23. 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

24. 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.

25. 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.