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

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


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: 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


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




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


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


Luminex xTAG RVPv1Virus % Sensitivity

Adenovirus 74.3

Influenza A 100

Infuenza A H1/09 100

Influenza A H3 92.9

Influenza B 95.5






Respiratory syncytial virus B 92.9



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


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


Parainfluenza virus 1 N/A




Respiratory syncytial virus B 85.7


• Pros– Quick time-to-result– High throughput

• Cons– No parainfluenza

coverage– Lower sensitivity


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.

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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