Clinical Microbiology Reviews-2018-Charlton-e00042-18.full

PRACTICAL GUIDANCE FOR
CLINICAL MICROBIOLOGY
crossm
Practical Guidance for Clinical Microbiology Laboratories:

Viruses Causing Acute Respiratory Tract Infections
Carmen L. Charlton,a,b Esther Babady,c,d,e Christine C. Ginocchio,f,g Todd F. Hatchette,h,i Robert C. Jerris,j Yan Li,k,l
Mike Loeffelholz,m Yvette S. McCarter,n,o Melissa B. Miller,p Susan Novak-Weekley,q Audrey N. Schuetz,r Yi-Wei Tang,d,s
Ray Widen,t Steven J. Drewsa,b
Downloaded from http://cmr.asm.org/ on March 7, 2020 by guest

a Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
b Provincial Laboratory for Public Health, Edmonton, Alberta, Canada
c Microbiology Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
d Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
e Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
f Zucker School of Medicine at Hofstra/Northwell, Hempstead, New York, USA
g bioMérieux/BioFire Diagnostics, New York, New York, USA
h Division of Microbiology, Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority, Halifax, Nova Scotia, Canada
i
Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
j Microbiology, Children’s Healthcare of Atlanta, Atlanta, Georgia, USA
k Influenza and Respiratory Viruses Section, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada
l
Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
m Department of Pathology, Clinical Microbiology Division, University of Texas Medical Branch, Galveston, Texas, USA
n Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Jacksonville, Florida, USA
o Clinical Microbiology Laboratory, UF Health-Jacksonville, Jacksonville, Florida, USA
p Department of Pathology & Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
q Medical Affairs, Qvella Corporation, Carlsbad, California, USA
r Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine and Science, Rochester, Minnesota, USA
s Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, New York, USA
t Esoteric Testing/R&D Pathology Department, Tampa General Hospital, Tampa, Florida, USA
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
EPIDEMIOLOGY AND CLINICAL PRESENTATION OF ACUTE RESPIRATORY VIRAL
INFECTIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Circulation of Respiratory Viruses: a Global Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Acute respiratory infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Mechanisms of transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Acute respiratory viral infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
(i) The host. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
(ii) Environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Citation Charlton CL, Babady E, Ginocchio CC,
(iii) Anatomic site of infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Hatchette TF, Jerris RC, Li Y, Loeffelholz M,
Zoonotic viruses: human-animal health interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 McCarter YS, Miller MB, Novak-Weekley S,
Section Summary and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Schuetz AN, Tang Y-W, Widen R, Drews SJ.
GUIDELINES ADDRESSING THE DIAGNOSIS AND MANAGEMENT OF SYNDROMES 2019. Practical guidance for clinical
ASSOCIATED WITH ACUTE RESPIRATORY INFECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 microbiology laboratories: viruses causing
Infectious Diseases Society of America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 acute respiratory tract infections. Clin Microbiol
Community-acquired pneumonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Rev 32:e00042-18. https://doi.org/10.1128/CMR
FLU-specific guidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 .00042-18.
Rhinosinusitis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
(continued) Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Steven J. Drews,
[email protected].
Published 12 December 2018
January 2019 Volume 32 Issue 1 e00042-18 Clinical Microbiology Reviews cmr.asm.org 1

Charlton et al. Clinical Microbiology Reviews
Other U.S. and International Guidelines Concerning Specific Populations and

Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
SOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
HSC recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Patients in the ED setting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Patients requiring isolation precautions in a health care setting. . . . . . . . . . . . . . . . . . . . . . . 13
Outbreak investigations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Emerging pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
(i) MERS-CoV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
(ii) Novel and emerging FLU strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Acute Respiratory Viral Infection following Travel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Section Summary and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
SPECIMEN COLLECTION FOR LABORATORY DETECTION OF ACUTE RESPIRATORY
VIRUSES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Risk Assessment for Emerging Pathogens Prior to Specimen Collection . . . . . . . . . . . . . . . . 15

Appropriate Specimen Collection Is Critical for Virus Detection in the Laboratory . . . . . . 15
When to collect a specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Biosafety considerations and PPE required for collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Sampling from upper respiratory tract sites: which specimen to use? . . . . . . . . . . . . . . . . 16
Approaches to specimen collection from the lower respiratory tract. . . . . . . . . . . . . . . . . . 17
Transport medium and transport considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
LABORATORY DETECTION OF ACUTE RESPIRATORY VIRUSES . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
The Role of Cell Culture is Limited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Direct Fluorescent-Antibody and Immunofluorescent-Antibody Assays for Respiratory
Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Rapid Antigen Detection Tests for the Detection of Respiratory Viruses . . . . . . . . . . . . . . . . 19
Molecular Detection Approaches as the New Reference Standard . . . . . . . . . . . . . . . . . . . . . . . 21
Extraction considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Assay control considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Positive predictive value and false-positive tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Labor and cost of molecular assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Understanding Applications of Molecular Detection Approaches. . . . . . . . . . . . . . . . . . . . . . . . . 24
Limited role of viral loads in predicting patient outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Molecular panel testing for respiratory viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
(i) Defining multiplex assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
(ii) Recommendations for patient populations in which multiplexed respiratory
viral panel testing may be appropriate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
(iii) To multiplex or not to multiplex? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
(iv) Near-patient or POC tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Appropriate Test Utilization in the Era of Molecular Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Stakeholder engagement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Choosing the right test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Recent Issues Surrounding LDTs for the Diagnosis of Acute Respiratory
Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
ANTIVIRAL AND PROPHYLACTIC AGENTS: IMPACT ON THE CLINICAL LABORATORY. 31
RSV Prophylaxis and Antiviral Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Treatment and Prevention of Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Relevance of FLU Antiviral Resistance Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CODING AND REIMBURSEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
SUMMARY Respiratory viral infections are associated with a wide range of acute
syndromes and infectious disease processes in children and adults worldwide. Many
viruses are implicated in these infections, and these viruses are spread largely via re-
spiratory means between humans but also occasionally from animals to humans.
This article is an American Society for Microbiology (ASM)-sponsored Practical Guid-
ance for Clinical Microbiology (PGCM) document identifying best practices for diag-
nosis and characterization of viruses that cause acute respiratory infections and re-
places the most recent prior version of the ASM-sponsored Cumitech 21 document,
Laboratory Diagnosis of Viral Respiratory Disease, published in 1986. The scope of the
January 2019 Volume 32 Issue 1 e00042-18 cmr.asm.org 2

Guidance: Acute Respiratory Tract Viral Infections Clinical Microbiology Reviews
original document was quite broad, with an emphasis on clinical diagnosis of a wide
variety of infectious agents and laboratory focus on antigen detection and viral cul-
ture. The new PGCM document is designed to be used by laboratorians in a wide
variety of diagnostic and public health microbiology/virology laboratory settings
worldwide. The article provides guidance to a rapidly changing field of diagnostics
and outlines the epidemiology and clinical impact of acute respiratory viral infec-
tions, including preferred methods of specimen collection and current methods for
diagnosis and characterization of viral pathogens causing acute respiratory tract in-
fections. Compared to the case in 1986, molecular techniques are now the preferred
diagnostic approaches for the detection of acute respiratory viruses, and they allow
for automation, high-throughput workflows, and near-patient testing. These changes
require quality assurance programs to prevent laboratory contamination as well as
strong preanalytical screening approaches to utilize laboratory resources appropri-

ately. Appropriate guidance from laboratorians to stakeholders will allow for appro-
priate specimen collection, as well as correct test ordering that will quickly identify
highly transmissible emerging pathogens.
KEYWORDS clinical, guidance, laboratory, respiratory, virus
INTRODUCTION
Background
The most recent version of the American Society for Microbiology (ASM)-sponsored
Cumitech 21 document, Laboratory Diagnosis of Viral Respiratory Disease, was published
in 1986 (1). The scope of the original document was quite broad, with an emphasis on
clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen
detection and viral culture. The date of publication of the most recent Cumitech
document was roughly 3 years after Kary Mullis’ initial work on PCR technology. Since
that time, the practice of clinical microbiology has significantly changed, most notably
with the development of molecular approaches that have increasingly replaced tradi-
tional methods for diagnosis of respiratory viruses. Specimen collection techniques
have likewise improved and have enhanced the predictive values of these new mo-
lecular methods. Development of electronic order entry systems, computerized labo-
ratory information systems, and automated reporting has reduced turnaround times
(TATs) for laboratory results dramatically even in environments where laboratory
centralization has occurred. The continual emergence of new respiratory pathogens
requires laboratorians to recognize laboratory testing limitations and understand when
and how to refer suspicious cases to public health reference laboratories.
Purpose
This document is an ASM-sponsored Practical Guidance for Clinical Microbiology
(PGCM) identifying best practices for diagnosis and characterization of viruses that
cause acute respiratory infections (ARIs). The document is designed to be used by
laboratorians in a wide variety of diagnostic and public health microbiology/virology
laboratory settings, especially by members of the ASM worldwide. As such, this
consensus document is structured to cover a wide range of practice settings, and to
reflect changes in available technology, clinical practice, and viral pathogens since
1986. The document outlines the epidemiology and clinical impact of acute respiratory
viral infections, including preferred methods of specimen collection and current meth-
ods for diagnosis and characterization of viral pathogens causing acute respiratory tract
infections. Laboratory-developed and commercial diagnostic tools, approaches for
diagnosis of emerging pathogens, and detection of antiviral resistance in influenza A
virus (FLUA) and influenza A virus (FLUB) infections are also discussed. Specimen
handling approaches for specimens from multiple body sites, such as nasopharyngeal
swabs (NPS), nasopharyngeal aspirates (NPA), nasal swabs (NS), nasal washes (NW),
oropharyngeal and throat swabs (OPS/TS), sputa, bronchoalveolar lavage (BAL) fluids,
bronchoalveolar washes (BAW), and other lower respiratory tract specimens, are cov-

ered. Given the changes in turnaround time for these newer technologies and increases
in clinical use, the document also addresses appropriate laboratory utilization of
diagnostic respiratory viral testing. The scope of the document has shifted since the last
version of the Cumitech, which included discussion on clinical overlap of viral patho-
gens causing acute respiratory tract infections as well as other pathogens that were
shown to infect the respiratory tract, such as atypical bacterial pathogens. The current
document focuses strictly on viruses that primarily cause acute respiratory infections,
related syndromes, or disease processes. Viruses that can infect or shed from the
respiratory tract but lead chiefly to other presentations such as rash, vesicles, parotitis,
gastroenteritis, or mononucleosis-like syndromes (herpes simplex virus, varicella zoster
virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, measles virus, rubella virus,
mumps virus, bocavirus, and hantavirus) are not discussed in this document.
The primary focus of this document is pathogens with well-documented causal

effects for acute respiratory infections, namely influenza A virus (FLUA), influenza B virus
(FLUB), respiratory syncytial viruses (RSVs) A and B, respiratory enteroviruses (EVs),
rhinoviruses (RVs), respiratory adenoviruses (ADVs), human metapneumovirus (hMPV),
parainfluenza viruses (PIVs) 1 to 4, and coronaviruses (CoVs) (NL63, OC43, HKU-1, and
229E). The document also discusses the diagnosis and characterization of emerging
respiratory viral pathogens, including CoVs (causing Middle Eastern respiratory syn-
drome [MERS] and severe acute respiratory syndrome [SARS]) and novel FLU strains
arising from swine and avian sources.
EPIDEMIOLOGY AND CLINICAL PRESENTATION OF ACUTE RESPIRATORY VIRAL

INFECTIONS
Circulation of Respiratory Viruses: a Global Problem
The increased capacity for molecular diagnostics worldwide has enhanced our
understanding of global circulation patterns of respiratory viruses (2). From the clinical
laboratory perspective, understanding the circulation patterns of viruses will influence
the predictive value of respiratory virus testing and potentially the interpretation of
respiratory virus test results based on pretest probability (3). A number of geographic
regions now have well-established surveillance systems for FLU and occasionally other
respiratory viruses associated with acute illness (4–7). A complicated global viral
circulation pattern shows that some viruses maintain consistent seasonality, while
others vary extensively. In the Northern Hemisphere, RV and respiratory EVs typically
circulate in the late summer and early fall (autumn), while FLUA predictably peaks in
December or January (Fig. 1). PIV types, however, have varied circulation patterns with
seasonality depending on the subtype, and dominant types can change from year to
year (8). Although we can begin to predict patterns of respiratory virus circulation as
surveillance and detection capacities improve (9), viruses may be identified outside
their normal seasonal infection patterns due to patient activities, such as travel to
regions where the virus is currently circulating (10). Knowing the travel history com-
bined with active pathogen surveillance (e.g., identifying a patient who presents during
a North American summer with acute respiratory infection after travel to the Southern
Hemisphere where FLU or RSV is circulating) can help direct appropriate infection
prevention and control measures, as not all respiratory viruses require the same level
of patient isolation (11, 12).
Acute respiratory infections. Acute respiratory infections (ARIs) are among the most
common infections reported worldwide. In the 2013 global disease burden study
sponsored by the World Health Organization, respiratory infections were listed as the
leading cause of infectious disease and as being responsible for approximately 120
million disability-adjusted life years (DALYs) (a measure of the disease burden and its
impact on quality of life) (13). Lower respiratory tract infections (LRTIs) accounted for
greater than 90% of all DALYs, with approximately 35% of cases occurring in children
less than 5 years old (13, 14). The impact of respiratory infections on human health is
reflected in the large number of hospital and emergency room visits for both adults and
children (e.g., in the United States, there are 140,000 to 710,000 FLU-related hospital-


FIG 1 Circulation of common respiratory viruses in a large geographic area within the Northern Hemisphere. The data
represent all acute respiratory virus testing for multiple years in a population of 4.1 million patients, using a common testing
algorithm. The seasonality of viruses varies. (Generated by S. J. Drews and the ProvLab Alberta Laboratory Surveillance and
Informatics Team, 2016.)
izations per year), where respiratory viral infection is the most common reason to seek
medical care (15, 16).
Mechanisms of transmission. Respiratory viruses are transmitted primarily through
two mechanisms: (i) inhalation of infectious droplets and (ii) contact with contaminated
fomites. Aerosol transmission is the most common route of infection. Large (10 to
100 ␮m in diameter) aerosolized droplets can transmit viruses from the index case to
a new host in close proximity (ⱕ0.9 m), while small (⬍10 ␮m in diameter) aerosolized
droplets, produced during coughing or sneezing or through aerosol-generating pro-
cedures, can carry viral particles to new hosts several meters away (ⱖ1.8 m). Transmis-
sion via fomites from self-inoculation of the respiratory tract mucosa is the second most
common route of infection (17) (Table 1). Survivability and infectivity of viruses on
surfaces may vary from hours to days and depend on a number of viral and nonviral
factors. Nonenveloped viruses are more likely to cause infection via direct contact, as
they are more stable in the environment than enveloped viruses and are therefore
more likely to survive for extended periods outside the host (18). Animal and climato-
logical model systems suggest that respiratory virus (e.g., FLUA) transmission may also
be enhanced under specific environmental conditions, such as low temperature and
low humidity (19–21). It is important for laboratorians and clinicians to be aware of
likely transmission routes used by respiratory viruses in order to implement adequate
infection control practices, select appropriate specimen types, and safely perform
laboratory manipulations (Table 1) (22).

Charlton et al.
TABLE 1 Classification and characterization of common respiratory viruses

Large-droplet
Nucleic acid transmission Survival time as fomites Isolation precautions
Virus group(s) Family (reference[s]) Nucleic acid structurea Enveloped (reference) (reference[s])b (reference[s])c
Adenoviruses Adenoviridae (333, 334) Linear, nonsegmented dsDNA No Yes (11) 14–30 days (18) Contact, droplet (11, 335)
Coronaviruses Coronaviridae (333, 334) Linear, nonsegmented (⫹) ssRNA Yes Yes (11) 1 h–28 days (18, 336) Airborne, contact, droplet,
standardd (11, 337)
January 2019 Volume 32 Issue 1 e00042-18

Enteroviruses, rhinoviruses Picornaviridae (333, 334) Linear, nonsegmented (⫹) ssRNA No Yes (11) Limited data Droplet, standarde (11)
Influenza A/B viruses Orthomyxoviridae (333, 334) Linear, segmented (⫺) ssRNA Yes Yes (11) 5 min–7 days (18, 338–341) Droplet, standard,f ⫾
airborneg (11, 335)
Parainfluenza viruses 1–4 Paramyxoviridae (333, 334) Linear, nonsegmented (⫺) ssRNA Yes Limited data (333) 4–10 h (18, 342) Contact, standard (11, 335)
Human metapneumovirus Pneumoviridae (333) Linear, nonsegmented (⫺) ssRNA Yes Limited data (333) Limited data Contact, standard (11)
Respiratory syncytial viruses A/B Pneumoviridae (333) Linear, nonsegmented (⫺) ssRNA Yes Yes (343) 20 min–8 h (18, 344) Contact, standard (11, 335)
adsDNA, double-stranded DNA; ssRNA, single-stranded RNA.
bSurvival on dry surfaces or hands. Survival times are impacted by temperature, humidity, and type of surface. Nucleic acid has been detected.
cIsolation precautions can be found at: https://www.cdc.gov/infectioncontrol/pdf/guidelines/isolation-guidelines.pdf. Note the caveat regarding the use of droplet precautions when undertaking procedures likely to create
splashes or sprays.
dIsolation precautions vary by coronavirus type; the listed precautions are specific to SARS-CoV.
eRhinovirus isolation precautions.
fChanges in hemagglutinin and neuraminidase may impact transmission of influenza viruses; for up-to-date seasonal recommendations see https://www.cdc.gov/flu/professionals/infectioncontrol/healthcaresettings.htm.
gAirborne transmission may be possible in some cases.
cmr.asm.org 6
Clinical Microbiology Reviews

Close contact in living environments such as long-term care facilities facilitate

transmission to the elderly, who are often at higher risk for severe outcomes from
respiratory virus infections, such as pneumonia, acute-care hospitalization, and death.
In addition, illness may also occur in staff members. Challenges may arise because these
environments are not thought of as primary health care environments and may not
have infection control protocols that are as stringent as those in health care settings
(23, 24).
Similarly, pediatric day care settings are another transmission setting for exposure to
multiple respiratory viruses. A prospective cohort study from Washington State iden-
tified RSV, ADV, and RV as leading pathogens, with hMPV and CoV being less frequent,
in children in day care settings (25), and air sampling experiments have identified RSV
these settings (26). Children attending day care are at increased risk for respiratory
infections (all etiologies), especially at the start of entry into day care (27), and can be

a potential source of RSV infection for premature infants, who are at high risk of severe
compilations and outcomes (28).
Acute respiratory viral infections. There is significant overlap in clinical symptoms
associated with the different viruses causing respiratory illnesses (Table 2). The U.S.
Centers for Disease Control and Prevention (CDC) has established influenza-like illness
(ILI) criteria used for epidemiological surveillance to identify patients with likely influ-
enza infection (29, 30). These clinical criteria include cough, fever (temperature greater
than or equal to 100°F [37.8°C]), and/or sore throat and no identifiable cause other than
influenza (31); however, the specificity of these criteria is poor, as many other patients
with noninfluenza respiratory viruses can present similarly (32). In many cases, acute
respiratory infection (ARI) due to these viruses is indistinguishable from illness due to
bacteria on the basis of clinical presentation alone. Table 2 provides examples of
diseases and disorders that are caused by respiratory viral infection; however, this table
does not exclude the possibility that an unlisted virus may be the causative agent of a
disease or disorder.
(i) The host. The host response to viral infections relies on elements of both the
innate immunity and the adaptive immunity. Epithelial cells covering the mucosal
surface of the airway constitute the first physical barrier encountered by respiratory
viruses. Here, tight junctions connect the cells and provide a sealed environment,
preventing viral movement outside the respiratory tract. A layer of mucus overlays the
epithelial surface, and an upward directional movement of cilia effectively traps and
clears virus particles from the airway epithelium (33, 34). Binding and phagocytosis of
viruses result in production of several proinflammatory molecules, including interleu-
kins (e.g., interleukin-1␤ [IL-1␤] and IL-18), ␣/␤ defensins, collectins, type I interferons
alpha/beta, and immunoglobulin A (IgA), and attract natural killer cells. Upregulation of
this innate immune response limits local spread of the respiratory viruses (34) and
serves as the front-line defense prior to activation of the adaptive immune system.
In infants, the immune system is still developing. The lack of complete immune
memory, reduced innate and adaptive immunity, and physiological differences in
airways compared to those in adults (35) increase the susceptibility to viral infections
and disease severity (36). The immune response to respiratory viral infections may be
augmented by protective effects of passive antibodies transmitted in utero (37) and
other factors, including breastfeeding (38, 39). Reinfections with the same virus are not
uncommon, and disease severity as well as patient outcomes is dependent on multiple
factors, including viral genetic diversity and intrinsic/extrinsic patient factors (34,
40–42).
Individuals at increased risk for complications due to respiratory virus infections
include children, older adults (⬎65 years old), patients with underlying respiratory
conditions, and those with suppressed immune functions (e.g., transplant patients). In
patients with underlying respiratory conditions (e.g., chronic bronchitis, chronic ob-
structive asthma, chronic obstructive pulmonary disease [COPD], or emphysema), a
decrease (mucostasis) or increase (mucus hypersecretion) in the mucociliary escalator
function may lead to decreased clearance of viral pathogens and increased risk of

TABLE 2 Diseases and disorders associated with respiratory viral pathogensa
Virus(es) Respiratory diseases and disorders Comments Key references
ADV Pharyngitis, common cold, laryngitis, Main cause of pharyngitis in infants and children; types 4 and 7 caused (130, 236, 281,
Charlton et al.
bronchitis, bronchiolitis, pneumonia in military recruits; multiplex NAAT-based assays which 345–349)
pneumonia include ADV are available; latency and persistent shedding can
confound interpretation of qualitative tests
CoVs NL63, OC43, HKU1, 229E, Common cold, pharyngitis, Multiplex NAAT-based assays are available for the detection and (236, 348–356)
SARS-CoV, and MERS-CoV laryngitis, bronchitis, bronchiolitis, differentiation four genotypes of CoV (229E, OC43, NL63, and HKU1);
pneumonia, SARS, MERS genotypes such as SARS-CoV and MERS-CoV can be detected only by
NAAT, often at reference/public health laboratories
EV Bronchiolitis, bronchitis, common EV-D68 is associated with severe respiratory illness outbreaks in the (236, 357, 358)
cold, pharyngitis, pleurodynia USA (2014); multiplex NAAT-based assays which include EV are
available; as described in the text, many panels cannot differentiate

between EV and RV or detect all types of EV (e.g., EV D68); LDTs
have been utilized for typing
hMPV Bronchiolitis, common cold, hMPV infection is associated with a substantial burden of (236, 281, 348, 349,
laryngitis, bronchitis, pneumonia hospitalizations and outpatient visits among children throughout the 359, 360)
first 5 years of life, especially during the first year; elderly adults also
susceptible; multiplex NAAT-based assays are available for hMPV
detection; two groups and four subgroups of hMPV can be detected
and identified by molecular assays
RVs Bronchiolitis, bronchitis, common The leading pathogen causing common cold and the most common (236, 348, 349, 358,
cold, pharyngitis, pneumonia viral cause (8%) of pneumonia in adults in the USA; multiplex NAAT- 361–363)
based assays are available for RV detection; molecular assays are the
only method for detection of RV genotype C; some molecular panels
cannot distinguish between RV and EV
FLUA (including subtypes H1, Bronchitis, bronchiolitis, common Common pathogen of pneumonia in adults; multiplex NAAT-based (236, 281, 286, 348,
H3, and H5) and FLUB cold, influenza, laryngitis, assays are available for FLUA detection; on-demand and point-of- 349, 361,
pharyngitis, pneumonia care molecular tests are available; genotyping and subtyping can be 364–366)
done by molecular assays; clinical relevance of viral load
determination merits further investigation
PIVs 1–4 Bronchiolitis, bronchitis, common PIV 1 and PIV 3 are the most common types causing bronchitis; PIV 4 (236, 281, 349, 354)
cold, laryngitis, otitis media, has not been confirmed to be a definite pathogen in humans;
pharyngitis, pneumonia multiplex NAAT-based assays are available, including detection and
differentiation of PIVs 1–4
RSVA and -B Bronchiolitis, bronchitis, common Leading cause of bronchiolitis and common pathogen of pneumonia in (236, 348, 349,
cold, otitis media, pneumonia children; disease severity is significantly associated with viral load 367–369)
rather than RSV subgroup; multiplex NAAT-based assays are available
for RSV, and some of them provide subgroup information on RSVA
and RSVB
aAbbreviations: ADV, adenovirus; SARS, severe acute respiratory syndrome; MERS, Middle East respiratory syndrome; CoV, coronavirus; EV, enterovirus; hMPV, human metapneumovirus; RV, human rhinovirus; FLU, influenza
virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus; NAAT, nucleic acid amplification test; LDT, laboratory-developed test.
cmr.asm.org 8

infection (33). In older adults, increased susceptibility to viral infections, age-dependent

vaccine effectiveness (43) and more severe disease have been attributed to waning
innate and adaptive immunity. Particularly, infection with RSV has been attributed to a
decrease in memory CD8⫹ T-cell function (44, 45). Similarly, immunosuppressed pa-
tients with profound and prolonged reduction in T-cell immunity are at increased risk
for severe disease from viral infection (particularly ADV, hMPV, PIV, and RSV infections)
(46, 47). A few studies have suggested that genetic polymorphisms of innate immune
effectors, such as Toll-like receptors (e.g., TLR-4), are associated with increased suscep-
tibility to severe respiratory viral infection (48, 49).
(ii) Environmental factors. Environmental factors may also influence the inci-
dence of disease caused by respiratory viral infection either alone or with other
underlying factors such as asthma (50). These factors may include the number of
siblings in family, environmental smoking exposure (51), air pollution, climatic

conditions, or weather (52, 53).
(iii) Anatomic site of infection. As the name suggests, most acute upper respi-
ratory tract infections (URTIs) affect sites in the upper respiratory tract, including the
larynx, nasal cavities, nasopharynx, oropharynx, throat, sinuses, conjunctiva, and
inner ear, and commonly manifest as rhinosinusitis or the “common cold” (54),
acute sinusitis (55, 56), acute laryngitis (57–59), conjunctivitis (54, 60–65), and otitis
media (64, 66, 67). (Table 2).
Viruses in lower respiratory tract infections (LRTIs) affect deeper structures below the
larynx, including the trachea, bronchus, and bronchoalveolar site, and manifest as
bronchiolitis (68–71), bronchitis (72–76), and acute pneumonia (77–81).
Zoonotic viruses: human-animal health interfaces. The One Health concept is an
integrative and collaborative approach that works to improve the health of humans
and nonhuman animals while ensuring the protection of the natural environment (82).
Clinicians and laboratorians should remain aware of the potential impact of One Health
human-animal interfaces to allow for the emergence of new human respiratory viral
pathogens (83, 84). Recent examples include human infection with the Middle East
respiratory syndrome coronavirus (MERS-CoV) with camel exposure (83), swine variants
of FLUA (84), pandemic FLU (pdm09), avian FLUA (e.g., H7N9) (85), and severe acute
respiratory syndrome coronavirus (SARS-CoV) associated with bats and civet cats (86,
87). Laboratorians should establish effective communication links with epidemiologists,
clinicians, and animal health experts to understand the impact of zoonotic viruses on
human illness (88). Identification of at-risk patients early by clinicians can reduce the
potential for nosocomial transmission of zoonotic pathogens. From the laboratory
perspective, this means following the epidemiology of emerging infections and com-
municating with clinicians and public health workers to assess risk and determine the
testing required based on travel histories and animal exposures (89–91). These ap-
proaches not only will identify patients at risk and allow public health practitioners to
implement strategies to reduce transmission and limit further exposure in health care
facilities and the community but also will ensure that laboratories can work up
specimens using appropriate biocontainment approaches to reduce the risk of labo-
ratory transmission of pathogens (92).
Section Summary and Recommendations

Respiratory viruses are a global problem with varied temporal and geographic
patterns of circulation. Laboratorians and clinicians should understand that multiple
viruses can cause similar signs and symptoms when infecting the upper or lower
respiratory tract. Although some viruses may be more likely to be associated with some
diseases, it is difficult to use clinical presentations alone to determine the causative
agent. Laboratorians should have a firm understanding of viruses that are circulating in
their region, as well as emerging infections in other regions of the world, as this
information may guide clinicians and laboratorians in developing appropriate algo-
rithms to test for agents causing respiratory illness.

GUIDELINES ADDRESSING THE DIAGNOSIS AND MANAGEMENT OF SYNDROMES

ASSOCIATED WITH ACUTE RESPIRATORY INFECTIONS
Laboratorians must consider how laboratory testing impacts the diagnosis and
management (including infection control considerations, treatment, and prophylaxis)
of patients presenting with ARIs so that they collaborate with their health care
providers to develop effective utilization strategies and develop algorithms that prior-
itize of testing of patients for whom results can influence clinical decision making. The
following section summarizes U.S. and international guidelines written in the English
language for the diagnosis and management of respiratory virus infections. Although
viral diagnosis does not typically affect the patient management of otherwise-healthy
adult patients, these guidelines identify scenarios where respiratory virus testing has
been identified to influence patient management.

Infectious Diseases Society of America
Community-acquired pneumonia. Together, the Infectious Diseases Society of
America (IDSA) and the American Thoracic Society (ATS) published consensus guide-
lines for the management of community-acquired pneumonia in adults in 2007 (note
that revisions of the ATS guidelines are in progress) (79). In the guidelines, they outline
specific microbiological testing recommendations and discuss how to take an appro-
priate travel history to support the diagnosis of pneumonia. The document identifies
respiratory viruses as an important cause of community-acquired pneumonia (CAP) in
outpatients and inpatients and emphasizes the importance of testing for and public
health reporting of emerging or novel virus strains. Improvements to diagnostic testing
using molecular approaches are encouraged, and drawbacks to rapid antigen testing,
including cost and false-negative and false-positive results, are discussed. The docu-
ment also provides support for use of antivirals (oseltamivir, zanamivir, or peramivir) in
the treatment of seasonal and pandemic FLU, and it strongly supports vaccination in
the prevention of seasonal influenza disease (79).
More recently (2011), the IDSA and the Pediatric Infectious Diseases Society (PIDS)
published combined guidelines for the management of CAP in infants and children
older than three months (93, 94). Since viral pathogens cause the majority of CAP in
preschool-aged children, antibiotic therapy is not routinely required in this population.
Testing for respiratory viral infections with a rapid, highly sensitive, and specific assay
is recommended, as it may reduce the use of antibiotics in patients without clinical,
laboratory, or radiological findings suggestive of bacterial coinfection. Antiviral therapy
should be started as early as possible in children with moderate to severe CAP when
FLU is circulating and symptoms are worsening. The group suggested that treatment
not be delayed for laboratory confirmation, as negative laboratory tests (especially with
rapid antigen testing) may not exclude disease. The American Academy of Pediatrics, in
a policy statement by the Committee on Infectious Diseases and Bronchiolitis, did not
recommend ribavirin for the treatment of RSV-CAP in infants. However, palivizumab
prophylaxis of RSV was recommended by the American Academy of Pediatrics (94). The
palivizumab guidelines have since been updated (95) and do not emphasize laboratory
testing for RSV. No recommendations were provided for the use of antivirals against
PIVs, ADVs, hMPVs, or CoVs in pediatric CAP.
FLU-specific guidance. In 2009, the IDSA released guidelines on the diagnosis,
institutional outbreak management, chemoprophylaxis, and treatment of FLU in adults
and children (96) (an update for this document is currently in process). Specific
demographic criteria were outlined for whom should be tested for FLU, and testing was
recommended only if results would influence clinical management. These situations
partially include the following: immunocompetent outpatients with acute febrile respi-
ratory symptoms (within 5 days of onset) at high risk for hospitalization or death,
immunocompromised outpatients with febrile respiratory symptoms (regardless of
onset date), and immunocompetent and immunocompromised hospitalized patients
with fever and respiratory symptoms, including CAP patients (regardless of onset date).
FLU testing was also recommended for elderly and infant patients with fever of

unknown origin or sepsis (regardless of onset date), children presenting for medical
care with fever and respiratory symptoms (regardless of onset date), patients who after
admission develop fever and respiratory symptoms (regardless of onset date), and
individuals (e.g., health care workers, residents, or visitors) with febrile respiratory
symptoms (within 5 days of onset) connected to an institutional FLU outbreak.
Rhinosinusitis. The IDSA “Clinical Practice Guideline for Acute Bacterial Rhinosinusitis
in Children and Adults” provides guidance on clinical presentations to identify patients
with viral and bacterial rhinosinusitis (97). Bacterial rhinosinusitis is defined as any of
the following (i) ⬎10 days of symptoms without improvement and with onset of high
fever (ⱖ102°F [39°C], (ii) high fever with purulent nasal discharge or facial pain during
the first 3 to 4 days of illness, or (iii) worsening symptoms (e.g., fever, headache, or
increase in nasal discharge) after apparent resolution of an upper respiratory tract
infection. This document emphasized the use of clinical approaches and not laboratory

testing to distinguish between bacterial and viral rhinosinusitis due to the self- limiting
nature of this illness (97).
Other U.S. and International Guidelines Concerning Specific Populations and

Settings
SOT. In 2013, the Infectious Diseases Community of Practice of the American Society
of Transplantation, the American Society of Transplantation, and the Canadian Society
of Transplantation released guidelines for infectious disease testing on solid organ
transplant (SOT) patients (98). The guidelines recommend testing for common respi-
ratory viral infections, including FLU, RSV, PIV, hMPV, RV, and CoV (99) with nasopha-
ryngeal swabs, nasal washes, or aspirates. The use of BAL fluid samples should be
considered for patients with negative upper respiratory tract specimens or with clinical
or radiological evidence of lower tract disease processes. Multiple approaches may be
used for diagnosis (e.g., nucleic acid amplification tests [NAATs], direct fluorescent-
antibody [DFA] tests, rapid antigen detection, or culture), but the guidelines emphasize
that NAAT is the most sensitive approach, and use of multiplexed NAAT improves the
diagnostic capacity by testing for a variety of targets, which should be seriously
considered in lung transplant patients. Prophylactic interventions for FLU (vaccination
and neuraminidase [NA] inhibitors [NAIs]) and RSV (palivizumab) and the use of
therapeutics for influenza (neuraminidase inhibitors) and RSV (ribavirin/intravenous
immunoglobulin [IVIG]) are also outlined in the document (99).
The American Society for Transplantation Infectious Diseases guidelines for the
diagnosis and management of ADV in solid organ transplant patients were published
in 2013 (100). The document describes posttransplantation timelines for risk of ADV
infection, where the first three months following SOT represents the highest risk. The
guidelines emphasize that pediatric patients had the highest incidence of ADV infec-
tion, at 6.25%, which carried an organ-specific risk level (liver ⬎ heart ⬎ kidney). In
adult SOT recipients (liver, heart, kidney, and kidney-pancreas), 10.5% of those with
self-limited viremia after transplant later developed ADV-associated respiratory symp-
toms within the first year. Although ADV subgrouping does not play a role in the clinical
laboratory, it may provide a sense of molecular epidemiology. For example, respiratory
tract infections were associated with subgroups B1 (serotypes 3, 7, 16, 21, and 50), B2
(serotypes 11, 14, 34, and 35), C (serotypes 1, 2, 5, 6), and E (serotype 4), while
disseminated disease (involvement of two or more organs) was associated with sub-
groups A (serotype 31), B2 (serotypes 11, 34, and 35), C (serotypes 1, 2, and 5), and F
(serotype 40). Multiple diagnostic approaches can be used for suspected ADV infection,
including NAAT, culture, DFA testing, and histopathology (considered the gold stan-
dard by the guidelines group for invasive ADV infection), but due to long-term
shedding in respiratory specimens (as well as urine and stool), detection of ADV is not
necessarily indicative of a disease process cause by ADV. Clinical symptoms, detection
of the virus in multiple sites, and histopathology may strengthen the association of ADV
detection with disease; however, the American Society for Transplantation Infectious
Diseases guidelines do not offer predictive algorithms to link detection of ADV in

multiple sites with disease. The lack of clear clinical cutoffs in qualitative and quanti-
tative NAATs adds to the confusion of whether positive results represent a current
active infection. Issues with false-negative ADV results with some NAAT panels are also
described later in this review. The American Society for Transplantation Infectious
Diseases guidelines indicate that NAAT on a blood sample may be used successfully to
monitor therapy, particularly if a baseline quantitative value is determined. ADV infec-
tions can be treated with cidofovir; however ribavirin should not be routinely used to
treat ADV infections even though some subtype C viruses may respond to ribavirin
treatment (100).
HSC recipients. International guidelines (combined recommendations of the Center
for International Blood and Marrow Transplant Research [CIBMTR], the National Marrow
Donor Program [NMDP], the European Blood and Marrow Transplant Group [EBMT], the
American Society of Blood and Marrow Transplantation [ASBMT], the Canadian Blood

and Marrow Transplant Group [CBMTG], the IDSA, the Society for Healthcare Epidemi-
ology of America [SHEA], the Association of Medical Microbiology and Infectious
Diseases Canada [AMMI], and the [CDC]) for preventing infectious complications in
hematopoietic cell transplant recipients were released in 2009 (101). Patients are at risk
from respiratory virus infection (FLU, RSV, hMPV, and PIVs) at all transplant stages from
preengraftment to late phase. Prolonged shedding times after viral infections were
identified in hematopoietic stem cell (HSC) recipients, with the following potential
shedding times for the following viruses: ADV, ⱖ2 years; FLU, ⱖ4 months; and RSV,
ⱖ22 days. Preventative measures for FLU include vaccination of close contacts and
antiviral prophylaxis (for close contacts and patients). No recommendations were made
for the use of ribavirin as a preemptive therapy for RSV. Evidence supporting the
efficacy of palivizumab prophylaxis for RSV prevention in HSC recipients ⬍4 years of
age was thought to be insufficient to recommend for or against use. No recommen-
dations were made for prophylaxis of PIV or hMPV infections. Testing for RSV and FLU
in HSC recipients with signs and symptoms of respiratory infection during periods of
circulation was recommended; however, routine surveillance of asymptomatic patients
for these respiratory viruses was not endorsed (101).
Recently, guidelines from the Fourth European Conference on Infections in Leuke-
mia addressed the diagnosis and treatment of RSV, PIVs, hMPV, RVs, and CoVs in
patients with leukemia and those undergoing HSC transplants (102). The group had
several recommendations regarding diagnosis of upper and lower tract community-
acquired respiratory viruses, including (i) testing to guide infection prevention and
control, treatment, and decisions for deferral of chemotherapy or HSC transplant, (ii)
evidence for collecting specimens from the site of involvement (e.g., pooled swabs for
the upper respiratory tract and BAL fluid [or tracheal swab if BAL fluid not available] for
the lower tract), (iii) evidence to support the use of first-line or routine diagnostic tests
for FLU, RSV, and PIV, (iv) evidence to test for other community-associated respiratory
viruses based on assessment of risk of exposure and local epidemiology, and (v)
evidence to consider collection of BAL fluid or biopsy samples for broader respiratory
viral pathogen testing in patients with lower tract disease. Treatment with ribavirin and
IVIG was recommended for RSV infection, while ribavirin alone was recommended for
patients with PIV infection (102).
In 2016, the Infectious Diseases Working Party of the German Society for Hematol-
ogy and Medical Oncology released guidelines for the diagnosis and management of
community-acquired respiratory viruses (103). The risk of infection with FLU, RSV, PIVs,
hMPV, and ADV in cancer patients is significant, and infection is associated with high
rates of pneumonia and mortality. The document highly recommends NAAT for RSV,
FLU, PIV, and other circulating/prevalent viruses in symptomatic patients. NAAT is
recommended over antigen detection or culture as the test of choice for identifying
these viruses. For patients with lower tract infection or critical illness, expanded testing
for hMPV and ADV (and potentially other rare causes of lower tract disease [e.g., RVs
and CoVs]) is suggested. Moderate support for recommendations for causal treatment
of FLU (oseltamivir, zanamivir, and peramivir), RSV (ribavirin and IVIG), and ADV

(cidofovir) was given. Marginal support for recommendations for causal treatments of
PIVs (ribavirin) was given (103).
Patients in the ED setting. In 2016, the American Academy of Emergency Medicine
approved a clinical practice paper for the vaccination, diagnosis, and treatment of FLU.
For seasonal FLU in the emergency department (ED), providers should (104) (i) perform
testing only if results will change clinical management, (ii) understand the limited
sensitivity and false-negative rates of rapid antigen detection tests (RADTs), (iii) con-
sider NAAT if clinical suspicion is moderate to high, and (iv) if rapid antigen detection
tests are negative but clinical suspicion is high, consider empirical antiviral therapy.
Additionally, FLU antivirals are recommended for patients who are (i) hospitalized, (ii)
at higher risk for complications, and (iii) have progressive illness (104).
Patients requiring isolation precautions in a health care setting. The Health Care
Infection Control Practices Advisory Committee (HICPAC) document “Guideline for

Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings”
discusses key functions of the clinical laboratory (11). The document recommends that
microbiologists help guide the limited application of rapid testing to clinical situations
where this testing influences patient management decisions and that they oversee
nonlaboratory workers who perform this testing. The document also recommends the
application of rapid tests to support treatment decisions, bed management, and
implementation of infection prevention and control measures (e.g., barrier precautions,
chemoprophylaxis, and vaccination); however, the authors of this PGCM document
emphasize that the test characteristics (e.g., sensitivity, specificity, and predictive
values) of an assay should be taken into account when making this decision. Surveil-
lance of FLUA and RSV was emphasized for case finding or cluster analysis, particularly
when infection precautions may be implemented. Removing a patient from isolation is
virus specific (see Table 1 regarding isolation precautions); however, of note, RSV
antigen tests are considered inadequate to remove patients from contact precautions,
as false-negative results are frequent.
Outbreak investigations. The U.S. CDC guidelines “Unexplained Respiratory Disease
Outbreaks (URDO)” outline the steps taken to define and investigate a respiratory
outbreak of unknown origin (105). Detection and characterization of the pathogen are
key steps allowing for effective clinical management, infection prevention and control
practices, and defining the time period of the outbreak. The document identifies a
variety of testing, including NAAT, culture, serology, and antigen detection, that may be
used to investigate the etiology of an outbreak (105).
Emerging pathogens. In the last few years a number of emerging viruses have been
identified globally, including FLU subtypes (H5N1, H5N6, and H7N9 [106–108]) and CoV
strains (MERS-CoV [109]\and SARS-CoV [110]). A number of guidelines have been
published to help in the diagnosis and management of these emerging pathogens
(111–113). Optimal timing of collection differs. Although the ideal specimen collection
time for influenza virus is as soon as possible after symptom onset, NAAT for MERS-CoV
can be performed 14 days postonset due to improved sensitivity of the assays. From the
laboratory perspective, NAAT is the recommended method of detection. A wide variety
of respiratory specimens may also be collected. If upper tract swabs are negative, then
lower tract specimen collection should be pursued. Although the cultivation of these
pathogens requires a higher level of biocontainment, the majority of activities for
identification via NAAT can be done in biosafety level 2 (BSL-2) facility in a biosafety
cabinet (BSC) using enhanced precautions. As new pathogens emerge (e.g., H7N4),
laboratorians should confer with reference centers (e.g., the U.S. CDC) on the most
appropriate testing approaches to detect and characterize these viruses.
(i) MERS-CoV. In June 2015, the most recent version of the MERS-CoV biosafety
guideline was released as “Interim Laboratory Biosafety Guidelines for Handling and
Processing Specimens Associated with Middle East Respiratory Syndrome Coronavirus
(MERS-CoV)—Version 2” (113). Activities appropriate for BSL-2 facilities using standard
BSL-2 practices included molecular testing of extracted nucleic acid and final packing
of specimens for transport to diagnostic laboratories for additional testing. Activities to

be undertaken in a class II BSC included aliquoting specimens, diluting specimens,

performing diagnostic tests not involving propagation of potentially infected speci-
mens, and nucleic acid extraction from potentially infectious specimens. Cell culture
propagation and the characterization of propagated material should be undertaken in
a BSL-3 facility using BSL-3 practices (113).
In June 2015, “Interim Guidelines for Collecting, Handling, and Testing Clinical
Specimens from Patients Under Investigation (PUIs) for Middle East Respiratory Syn-
drome Coronavirus (MERS-CoV)—Version 2.1” was released by the CDC (114). The
guidelines recommended, when possible, the collection of upper respiratory tract,
lower respiratory tract, and serum specimens for the diagnosis of MERS-CoV. Potential
lower respiratory tract specimens included BAL fluid, tracheal aspirate, pleural fluid, and
sputum. Appropriate upper respiratory tract specimens included NPS and OPS (which
could be combined in the same transport container if the test is validated for this type

of combined collection) and nasopharyngeal aspirates. Upper and lower respiratory
tract specimens should be collected within 7 days of symptom onset; however, NAAT
can be performed 14 days postonset due to improved sensitivity of the assays (112).
(ii) Novel and emerging FLU strains. In January 2014, guidelines for possible infection
with avian FLUA (H7N9) virus were released by the CDC (111), and these were later updated
for novel FLU strains (116). These guidelines outline appropriate testing for emerging FLU
strains such as A(H7N9) and A(H5N1), and they describe exposure risk and clinical symp-
toms specific for each virus. Specimens should be collected as early as possible after
symptom onset (ideally within 7 days) (116). Sample collection after this point is still
relevant in children, immunocompromised patients, and critically ill patients with lower
tract disease, as virus can be shed for longer periods in these patient populations. As new
strains emerge (e.g., H7N4), laboratorians should confer with reference centers (e.g., the U.S.
CDC) on the most appropriate testing approaches to detect and characterize these viruses
(117).
Acute Respiratory Viral Infection following Travel

The book CDC Health Information for International Travel (also known as the “Yellow
Book”) (118) is a reference for health professionals who care for international travelers.
The “Yellow Book” identifies viral pathogens as the most common cause of respiratory
infections in travelers. Etiologies can vary widely, including infection with RV, RSV, FLU,
PIVs, hMPVs, ADV, or CoV (118); however, in the absence of severe illness or pneumonia,
laboratory diagnosis is not always clinically necessary (118). Depending on the travel
history, novel causes of respiratory illness (e.g., MERS-CoV and avian FLU strains) should
be considered for symptomatic patients.
It should be noted that the positive predictive value (30 to 88%) for laboratory-
confirmed influenza in returning travelers can vary widely depending on the seasonality
of infection and method of detection (119). While the negative predictive value of FLU
NAAT in returning travelers can be used to rule out FLU infection, earlier-generation
antigen detection test methods should not be used to rule out influenza virus infection,
particularly when emerging strains are suspected. Patients who should be tested for
FLU infection include (i) symptomatic hospitalized patients, (ii) cases where diagnosis of
FLU will affect patient management, and (iii) cases where FLU testing would affect
infection prevention and control or management of close contacts (119).

Multiple guideline groups have addressed the role of laboratory diagnosis of viruses
in specific patient populations. Laboratorians should be aware that many guidelines are
greater than 5 years of age and may not have taken into account the changes that have
occurred in the types of tests available for the diagnosis of respiratory viruses. Although
some of these documents are now aging, it is clear that testing may play a more
important role in the management of severely ill patients and the immunocompro-
mised and less of a role in the management of immunocompetent and relatively
healthy adults and children. Laboratory testing may assist in supporting public health

investigations (e.g., emerging pathogen investigations and outbreak investigations),

epidemiological investigations, and infection control functions. Most simply, laboratory
testing may be considered when it positively impacts clinical decision making and
supports patient management.
SPECIMEN COLLECTION FOR LABORATORY DETECTION OF ACUTE RESPIRATORY

VIRUSES
Risk Assessment for Emerging Pathogens Prior to Specimen Collection
During clinical assessment, clinicians should ask about travel history and animal
exposure that could be consistent with acquisition of (or exposure to) an emerging
pathogen (e.g., MERS-CoV or avian FLU). Prompt consideration of an emerging patho-
gen based on epidemiological risks with engagement of public health and the imple-
mentation of appropriate of infection prevention and control measures are essential to

prevent nosocomial spread of these infections. In the MERS-CoV outbreak in South
Korea (May to July 2015), the lack of prompt identification of risk factors in patients
presenting to the ED allowed spread between patients and staff at several hospitals
(120). Early identification and upfront screening procedures could have isolated the
index patient and reduced the number of contacts, thus limiting the spread of infection
(121). This is consistent with mathematical modeling showing that rapid identification
of index cases is the most important factor in reducing spread of infection and that
patient isolation and quarantine have the strongest correlation with transmission
prevention (122). As soon as an emerging pathogen is suspected, the laboratory should
be notified to provide advice on appropriate specimen collection and testing to ensure
identification and to ensure that the specimens are handled with the appropriate
biocontainment considerations for the novel pathogen.
Appropriate Specimen Collection Is Critical for Virus Detection in the Laboratory

When to collect a specimen. Clinicians should collect specimens from symptomatic
individuals with acute respiratory illness within 5 days of symptom onset (preferably
within 48 h). Specimen collection later than 5 days after onset is recommended only
when symptoms persist or worsen, in young children, or in the immunocompromised
(96, 123).
Virus-specific shedding estimates can further direct best collection guidelines for
respiratory specimens; however, it should be noted that estimates are typically per-
formed on select patient populations, and differences may be due to differences in
study designs, differences in specimen types, and differences in detection technologies
between studies (124–126). NAAT is the most sensitive method of detection, and
sampling as soon as possible after the onset of symptoms is considered ideal for
healthy individuals; most viral targets can be effectively identified in the first 2 days
after symptom onset, and multiple studies indicate that viral loads in respiratory
specimens will generally decrease over time. Furthermore, a delay in specimen collec-
tion following onset of respiratory symptoms will negatively impact the sensitivity of
laboratory tests to detect a pathogen.
In RV infection, NAAT identified peak shedding within 2 days of symptom onset,
with decreasing viral loads up to 7 days after onset (127). When virus culture and NAAT
were both used to test specimens, 57% of human hMPV isolates were detected within
the first 2 days of symptom onset, while only 19% were detected greater than 4 days
after onset (128). Only 27% of hMPV NAAT-positive specimens collected after day four
were positive by culture (128). In children tested for RSV by DFA testing, viral shedding
(measured in upper respiratory tract specimens [e.g., nasal, throat, and NPS specimens])
peaked 2 days after onset of illness, and the median shedding duration was 4.5 days.
Similar shedding patterns were identified for FLU infection. In community patients with
acute respiratory illness, FLUA viral loads measured by NAAT peaked at day one
following symptom onset and were detected until day eight, while in patients who had
one symptom (but did not meet the case definition for acute respiratory illness), loads
peaked on day one with detection until day six (129). In contrast, FLUB viral loads were

found to be highest on the day of symptom onset and to persist until day six to eight
(129).
It should be noted that there are no standard “case definitions” on how long positive
respiratory virus results detected by NAAT should be considered part of the same
infection event. Some preliminary studies propose a 30-day period for ADV infection in
children and for RV infection in infants as a definition of a single case (130, 131);
however, the temporal definition of a new viral infection should be assessed in the
clinical context, as the presence of comorbidities can significantly alter viral shedding
times. The duration of shedding can be influenced by multiple factors. Although prior
infection may not completely prevent reinfection, it may alter the duration of shedding.
Older individuals (suggested to have prior exposure) and children with prior RSV
infection generally shed for shorter periods of time (125, 132). The strain of virus or
subtype or coinfection with different viruses (133, 134) can also influence shedding

patterns. RSVA was detected 5.8 days longer than RSVB (135). Similarly, when children
with acute expiratory wheezing were found to be coinfected with EV and RV, shedding
of RV persisted for 2 to 3 weeks, whereas EV shedding persisted for 5 to 6 weeks (136).
Other factors may increase shedding time and still allow for productive specimen
sampling and detection of viral pathogens. Some studies suggest that viral shedding
may also be extended in patients with more severe disease (125, 137). Shedding can
also be prolonged in immunosuppressed patients. Although the number of patients
with detectable virus (FLU, PIV, or RSV) was highest in the first 2 weeks following
symptom onset, long-term virus detection (⬎30 days) with NAAT on upper and lower
respiratory tract specimens has been described for FLU, PIV, and RSV in patients with
hematological disorders (138). Testing these patients for “test of cure” is not recom-
mended or appropriate for viral upper respiratory tract infections, as viral shedding
often does not represent active infection (139).
Biosafety considerations and PPE required for collection. Respiratory viruses such
as FLU can be efficiently transmitted through the air (140, 141); however, the direct risk
to health care workers who are collecting upper and lower respiratory tract specimens
by different aerosol-generating procedural methods (e.g., bronchoscopy, sputum in-
duction, endotracheal intubation, positive pressure ventilation, nebulizer treatment,
airway suction, tracheostomy, chest physiotherapy, and high-frequency oscillatory
ventilation) is currently unknown (142). Analysis of historical data is confounded by
growing evidence that infection prevention and control practices for respiratory viruses
may not be uniformly followed (143). A recent analysis of practices in multiple U.S.
states found low practice adherence, with many health care workers unsure of when
appropriate personal protective equipment (PPE) should be worn (143). Droplet pre-
cautions for patients with confirmed or suspected infection with FLU should be
practiced to prevent transmission during collections. The need for N95 masks can be
controversial, and local infection prevention and control procedures should be fol-
lowed to minimize aerosolization and risk of health care worker infection (144–146).
Even if more “effective” respirators are used when clinicians are in contact with patients,
their benefit may be negated if generally poor infection prevention and control
practices are utilized (145). The laboratorian with expertise in respiratory virus trans-
mission and viral characteristics can be a valuable member of local teams when
creating respiratory protection program protocols.
Sampling from upper respiratory tract sites: which specimen to use? For an upper
respiratory tract infection, a variety of specimens can be used to diagnose respiratory
infections (NPA, NPS, NW, NS, OPS/TS, and sputum) (Table 3), and the U.S. CDC offers
collection guidance for each; however, laboratories should use manufacturers’ recom-
mended specimen types in U.S. Food and Drug Administration (FDA)-cleared or vali-
dated/verified laboratory tests (147, 148). Selection of specimen type is dependent on
a variety of factors: patient age, patient willingness to undergo a specific procedure,
clinical presentation, the nature of the potential pathogen, and the appropriateness of
the specimen type for verified laboratory diagnostic approaches. Although a combi-
nation of different specimen types can improve the sensitivity of NAATs (149–155), this

TABLE 3 Sensitivity of respiratory viral detection from different specimen typesa

Sensitivity of detectionb of:
Specimen type FLUA/Bc RSV RV/EV ADV hMPV PIVs CoVsc
NPS ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹
NPA ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
OPS ⫹⫹(⫹)d ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹
TS ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹
Sputumf ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹(⫹) ⫹⫹(⫹)e
BAL fluid ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹(⫹) ⫹⫹
Lung biopsy specimen ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ o ⫹⫹⫹
aFor specimen collection, it is important that appropriate infection control practices are followed, as
collection can be aerosol generating. FDA clearance and laboratory-based validation/verification of the
specimen source for assay need to be considered. Appropriate collection methods should consider
downstream testing to ensure that specimens are handled, stored, and shipped properly prior to testing.
Preanalytical specimen storage information provided by the laboratory should indicate storage temperature,

retention time, and stability of the specimens (123, 178, 179, 370). Combinations of different specimen
types can significantly increase the yield for viral detection. Results for nasal specimens are not included in
this table because the literature describing their efficacy in detection is variable (372–374).
b⫹⫹⫹, specimen type has high detection rates for the indicated virus; ⫹⫹, specimen type is acceptable for
viral detection, but sensitivity may be reduced due to the sampling or testing method used for detection;
⫹, specimen type has reduced sensitivity for indicated virus; ⫹⫹(⫹), minor reduction; ⫹(⫹), moderate
reduction; o, limited utility.
cFor emerging avian influenza virus strains or for CoVs such as SARS-CoV or MERS-CoV, lower respiratory
samples are additionally recommended for enhanced detection.

dNPS were more sensitive for detection of FLUB, while OPS were more sensitive for FLUA strains (153).
eSputum sensitivity varies between CoV strains (180).
fSensitivity of sputum results can vary widely depending on the quality of the specimen received. Sputa
received for viral testing are not screened for specimen adequacy as for those received for bacterial workup
(371).
must be balanced in such a way to maintain high detection rates yet still maintain a
cost-effective approach. For emerging pathogens (e.g., novel FLUA H5/H7/H9 or emerg-
ing CoV), a collection of multiple different types (OPS, NS, NPS, BAL fluid, etc.) may be
necessary to identify specimens that most reliably result in detection of the pathogen.
Depending on the pathogen (e.g., emerging CoV or novel FLUA), other, atypical
specimens such as blood or stool for direct virus detection may also be suggested for
collection (156–158).
Traditionally, NPA were used as the gold standard for detection of respiratory viruses
(159). Previous publications suggest that NPS is equivalent to NPA for the detection of
multiple viruses in children (160). Although NPS/NPA are generally more sensitive than
throat swabs for detection of most viruses (152, 154, 161, 162), NS are easier to obtain,
are less painful (163–165), and can be self-collected with yields equivalent to those
collected by a clinician (166). Reduced diagnostic sensitivity using NS samples is often
considered an acceptable trade-off for increased compliance, particularly when the
prevalence of disease is high (159, 167). In addition, there are increasing data suggest-
ing that the combination of both an NS and an OPS in adults and children has a yield
equivalent to that of NPS/NPA (10, 151, 155). Use of a flocked swab with a liquid viral
transport medium may additionally improve viral detection (161, 168, 169). Easier
midturbinate collection with flocked swabs may provide an alternative to proper
nasopharyngeal specimens, albeit with potentially a lowered sensitivity (170, 171).
Finally, when using commercially available rapid antigen detection tests (RADTs),
laboratories should use the kit-recommended swab unless the performance of the test
with a different specimen type has been verified (172).
Approaches to specimen collection from the lower respiratory tract. Lower respi-
ratory tract specimens such as sputum, bronchoalveolar lavage/wash, and lung tissue
may be considered in cases where the patient may be infected with an emerging
pathogen (173, 174) or is under intensive/critical care for pneumonia (175), in cases
involving autopsy (176), or where molecular detection requires pathological evidence
of invasive disease (e.g., ADV infection in lung specimens of lung transplant patients)
(177). In severe illness due to influenza and emerging pathogens, upper respiratory

tract sampling may yield false-negative results (112). Accurate diagnosis in these cases
often will require a variety of specimens from the upper and lower respiratory tracts.
Selection of lower respiratory tract specimens should be dependent on the disease
course (e.g., anatomic location of the diseases process, stability of the patient/risk in
sampling, and ability to access the anatomic site) (176, 178–180). Given these issues,
specimen collection and therefore determination of the lower respiratory tract infection
may not be possible.
Lower respiratory tract specimen types vary in their ability to be used to detect
specific viral etiologies (Table 3). Sputum may be considered an appropriate specimen
for sampling the lower respiratory tract in some patients (178–180). However, data are
limited. Specimen viscosity and higher rates of PCR inhibition make sputum a more
difficult specimen type to use in the laboratory (174), and most FDA-cleared assays for
respiratory viruses are not validated by the manufacturer for sputum or other lower

respiratory tract samples (e.g., BAL fluid). Bronchial washes and lavage fluids can be
useful specimen types, provided that they are collected appropriately in sterile con-
tainers, as the viral load for lower respiratory tract infections can be higher in these
specimen types. Lung tissue collected during bronchoscopy, open surgical procedure,
or autopsy should be placed in a sterile container with a small amount of sterile saline
to keep it moist (176). Specimens should not be put into formalin, as it reduces the
sensitivity of NAAT, and formalin-preserved samples are not commonly verified sample
types for most laboratory test systems. Procedural variability for specimen collection
(e.g., volumes collected and dilution factors) makes comparison of the performances of
these off-label specimens difficult.
Transport medium and transport considerations. Viral transport medium or uni-
versal transport medium facilitates viral culture, direct fluorescent-antibody (DFA)
testing (181), rapid antigen detection tests (RADTs) (182), and molecular testing (181,
183, 184). Stability guidelines outlined in the package insert (storage at room temper-
ature, refrigeration, or freezing) should be used as per the manufacturer’s instructions.
Other transport devices may be considered (e.g., dry swabs [185, 186] or alcohol-based
transport medium [185]) but are not widely used. Transport should be in accordance
with regulators’ guidelines in each jurisdiction.

Always ensure that clinicians are aware of processes for safe specimen collection
from patients who are suspected of being infected with routine and emerging respi-
ratory viruses. For the detection of routine seasonal respiratory viruses, samples should
be collected as early as possible from patients following onset of illness. Shedding
studies of multiple viruses indicate that viral titers drop daily following the onset of
illness. Thus, sampling from patients at later time points is expected to negatively
impact the sensitivity of diagnostic assays. Sample collection from the upper respiratory
tract may be easiest, but upper tract sampling may not detect viruses causing lower
tract disease. Following specimen collection, ensure that appropriate transport and
storage conditions are used for specimens.
LABORATORY DETECTION OF ACUTE RESPIRATORY VIRUSES

The Role of Cell Culture is Limited
Cell culture was long considered the gold standard for virus isolation and identifi-
cation prior to the availability of molecular assays (187, 188). Modification of cell lines
(including primary lines, immortalized lines, mixed cell lines, and transfected lines) has
improved the ability to detect respiratory viral pathogens (189). For laboratories
offering cell culture analyses, detailed procedures can be found in the Clinical and
Laboratory Standards Institute document “M41: Viral Culture.” (190).
There are a variety of drawbacks to using cell culture compared to molecular
methods, and many virology laboratories have opted to discontinue viral culture in the
laboratory for these reasons. It is well established that cell culture has a lower sensitivity
than molecular techniques (191, 192), the turnaround time and hands-on time required

to perform cell culture compared to molecular testing are increased, and the technical
expertise for performing cell culture is often not available. Traditional tube cultures are
slow and can take up to 10 days for detection of respiratory infections (36). A study in
pediatric patients indicated that positive viral culture results would not impact the
management of healthy children hospitalized for illness attributed to community-
acquired respiratory viral infection due to the delay in time to culture positivity (193).
Shell vial assays can decrease the time to detection; however, 1 to 2 days is still required
for growth and identification of the virus. Care must be taken when selecting cell lines
for viral growth, as not all cell lines will allow for propagation of all viruses, and cell lines
may be viral strain specific (194). Yields from cell culture are often decreased following
freezing, due to reduced numbers of viable virus particles; therefore, samples that are
frozen prior to culturing may be falsely negative (139, 195). As a safety note, culture
approaches may inadvertently propagate emerging pathogens and compromise labo-

ratory biosafety (195); however, maintaining cell culture capabilities in public health
laboratories remains important for identification of unknown or emerging pathogens,
particularly when specific molecular amplification processes are not available, and can
provide an understanding of the virus viability within a clinical specimen (196).
Direct Fluorescent-Antibody and Immunofluorescent-Antibody Assays for Respi-

ratory Viruses
Direct fluorescent-antibody (DFA) and immunofluorescent-antibody (IFA) assays
have been used to detect a variety of respiratory viruses from primary specimens
(chromatographic immunoassays for the detection of respiratory viruses are discussed
in the following section). Commercial and standardized clinical reagents are available
for select respiratory pathogens (e.g., FLUA/B, PIVs 1 to 3, ADV, hMPV, and RSV) (197).
Like that of traditional cell culture techniques, the quality of DFA/IFA assays is impacted
by specimen quality and collection method (171). Unlike the case for traditional cell
culture, DFA and IFA assays do not require viable viruses and the turnaround times are
short (⬍4 h) and on a single-specimen basis can be shorter than those for older
laboratory-developed molecular approaches (which have, e.g., separate extraction
steps, greater numbers of manual steps, and manual interpretation and data entry in
laboratory information systems) or batched-based testing; however, DFA/IFA technol-
ogies are labor-intensive, require a skilled technologist to read and interpret results,
require a fluorescence microscope, and are subject to reader error. Furthermore, the
hands-on time required per test is not structured for high-throughput result reporting.
Compared to molecular detection methods, DFA and IFA assays have significantly
reduced sensitivity and specificity (197). Some argue that the lower sensitivity can
identify “clinically relevant infections” in some patient populations (e.g., hospitalized
pediatric patients) (198) in contrast to detection of free nucleic acid as in molecular
detection. Additionally, microscopic examination of samples for DFA testing can di-
rectly determine specimen quality (199) by allowing for observation of the number of
epithelial cells present in the sample.
Rapid Antigen Detection Tests for the Detection of Respiratory Viruses

Clinical Laboratory Improvement Amendments (CLIA)-waived tests are intended for
use in “professional” settings (e.g., physicians’ offices, mobile clinics, and pharmacies)
and/or by untrained operators with no laboratory expertise (200). A summary of rapid
antigen detection test (RADT) technologies that may be used as near-patient or
point-of-care (POC) tests is given in Table 4. Technologies for these guidelines are
discussed in general here; specific products are not discussed, and company names are
not mentioned.
Earlier RADT assays detected antigens of FLUA, FLUB, and RSV. Use was often
restricted to specific specimen types (e.g., NPS or NS), the sensitivities of these assays
in pediatric and adult populations varied but were considered to be poor, and the
assays could not be used to rule out infection (201, 202). Performance characteristics of
these assays were typically determined during normal respiratory virus seasons, with

TABLE 4 Respiratory viral testing approaches with CLIA waiversa
Specificity (%) vs
Patient Viral Sensitivity (%) vs real- real-time PCR Specimen General
population(s) Virus(es) targets Technologies time PCR assays assays type(s) TAT References
Charlton et al.
Clinically ill, no FLUA, FLUB Antigens Immunochromatographic FLUA, 15–56; FLUB, 24–56; FLUA, 99–100; FLUB, NPS, NS, NW/ Minutes 207, 375–378
age (first generation) combined, 23–69 99–100; nasal aspirate
restrictions combined, 96–97
Clinically ill, RSV Antigens Immunochromatographic 58–80 91–100 NPS, NPA, NW/ Minutes 212, 375,
neonatal (first generation) nasopharyngeal 379–381
and wash
pediatric,
age varies
Clinically ill, no FLUA, FLUB Antigen Assisted/automated reading FLUA, 67–81; FLUB, 33–92 FLUA, 98–100; FLUB, NPS, NS, NW/ Minutes 211, 288, 376,

age of immunoassay (second 90–100 nasal aspirate 381, 382
restriction generation)
Clinically ill, RSV Antigen Assisted/automated reading 78–82 97–99 NPS, NW/nasal Minutes 214, 383
pediatric of immunoassay (second aspirate
patients, age generation)
varies
Clinically ill, no FLUA, FLUB Nucleic Isothermal amplification FLUA, 73– 94; FLUB, FLUA, 63–100; FLUB, Direct NS or NPS ⱕ1 h 286–288, 384
age acid and molecular beacon 75–97 54–100
restriction probe detection
Clinically ill FLUA, FLUB Nucleic Automated sample FLUA, 98–99; FLUB, FLU, 99–100; FLUB, NPS ⱕ1 h 289, 385
acid preparation, 99–100 99–100
amplification, detection,
and result interpretation,
real-time multiplex
RT-PCR
Clinically ill FLUA, FLUB, RSV Nucleic Automated sample FLUA, 95; FLUB, 100; RSV, FLUA, 98; FLUB, 99; NPS ⱕ1 h 386, 387
acid preparation, 99 RSV, 99
amplification, detection,
and result interpretation,
real-time multiplex
RT-PCR
Clinically ill FLUA H3 subtype and Nucleic Single-instrument 96.8% positive agreement 99.5% negative NPS Approx 388, 389
adult and 2009 H1 subtype, acid configuration, real-time vs other FDA-cleared agreement vs 1h
pediatric FLUB, ADV, CoV, multiplex NAAT molecular comparative other FDA-cleared
patients hMPV, PIV 1–3, method molecular
RSV, RV, EV comparative
method
aAbbreviations:FLU, influenza virus; RSV, respiratory syncytial virus; ADV, adenovirus; CoV, coronavirus; hMPV, human metapneumovirus; PIV, parainfluenza virus; RV, rhinovirus; EV, enterovirus; NAAT, nucleic acid
amplification test; RT-PCR, reverse transcription-PCR; NPS, nasopharyngeal swab; NPA, nasopharyngeal aspirate; NW, nasal wash.
cmr.asm.org 20

acceptable specificity for RSV and FLU (203, 204); however, the performance charac-
teristics are significantly reduced when assays are used out of season (205–207). Many
believe that the clinical utility of employing FLU and RSV POC assays, given the high
numbers of both false-positive and false-negative results, is questionable (205–207),
and the future long-term availability of rapid antigen detection kits is in doubt. On 23
February 2017, the U.S. Food and Drug Administration (FDA) reclassified rapid antigen
influenza virus test kits from class I to class II medical devices (208). This was meant to
address growing concern about the variable performance of these assays as well as
poor sensitivity compared to other methods such as NAATs and culture. Existing kits
could be purchased until 12 January 2018 and used until the kit expiry date. Following
that point in time, manufacturers were expected to monitor kit reliability and provide
updates to users. Additionally, some assays are unable to differentiate between FLUA
and FLUB, which may impact epidemiological investigations (209), and they have

particularly poor sensitivity to detect avian influenza virus and other emerging sub-
types (210). However, RADT may still have a place in management of outbreaks or in
locations with limited access to molecular diagnostics (209), but consideration of the
assay performance and the seasonality should be taken into account when using these
assays.
A second generation of viral antigen POC tests improved the sensitivity for FLUA/B
and RSV detection compared to that of earlier technologies (211, 212); however, the
performance characteristics were still reduced compared to those of routine molecular
testing (Table 4). Similar to the case for earlier generations, respiratory viral infection
could not be ruled out with the newer POC tests, and sensitivities and specificities
varied depending on the FLU target and the comparator molecular method used (Table
4). For RSV, sensitivity and specificity were reduced compared to those with molecular
methods (Table 4). The sensitivity of these tests is highest during the RSV season when
the positive predictive value is high (213–215). If clinicians feel there is a need for RSV
antigen-based POC testing for pediatric patients (e.g., when there is no nearby labo-
ratory access or in resource-poor environments), laboratorians need to inform clinicians
of the newer test technologies, provide information on the current prevalence of these
pathogens, and assist in generating algorithms that reduce the risks of these technol-
ogies.
Molecular Detection Approaches as the New Reference Standard

Extraction considerations. The first step in NAAT requires extraction, purification,
and preservation of target organism nucleic acids. Extraction technologies should be
able to cleanly isolate both high-quality viral RNA and DNA and, depending on the
assay, to additionally sample human nucleic acids to allow the detection of human
genes (e.g., that for glyceraldehyde 3-phosphate dehydrogenase [GAPDH] or ␤2-
microglobulin [␤2M]) as control targets. The ubiquitous presence of RNase enzymes in
most human samples makes isolation of RNA nucleic acid targets (e.g., FLU, RSV, and
CoV) (Table 1) more difficult than isolation of DNA (e.g., adenovirus) (Table 1) and often
requires additional steps for processing. Multiple extraction methods may be employed
for respiratory virus detection. Heat-mediated lysis is an approach where target organ-
isms are lysed or homogenized to release target nucleic acids (216). This approach is
used in some commercial NAATs. Manual extraction using phase separation, capture via
magnetic beads, or immobilized silica spin or vacuum wash columns may also be used.
Automated extraction systems may be employed and generally use magnetic silica or
other particles designed to capture RNA, DNA, or both. In fully automated instrument
systems, all steps from extraction through to amplification are incorporated into a
single cartridge or pouch.
Commercially available respiratory virus NAAT kits for detection of respiratory viral
targets generally have a specific extraction method that is qualified for sample pro-
cessing as part of the FDA clearance. Often, the FDA-cleared NAATs will have claims for
specific specimen types (NPS, NS, etc.) but may or may not specify the type of transport
medium. If the laboratory chooses to use specimen types besides those that are FDA

cleared, the laboratory should perform a verification study to document recovery of the
target nucleic acids and acceptable performance of the NAAT (217). A validation plan
should consider a variety of factors, including the frequency of specimen type being
tested and the risk that specimen types may not be compatible with the assay.
Similarly, if the testing laboratory chooses to use a different extraction protocol,
verification for comparable performance is required. The requirement for verification of
additional specimen types is outlined in the College of American Pathologists’ Micro-
biology Checklist, Molecular Microbiology, MIC.64810 (sections titled “Test perfor-
mance—manufacturer’s instructions” and “Laboratory-developed or modified FDA-
cleared/approved tests”) (217). Many in-depth documents and reviews discussing the
requirements of molecular assay validation have previously been published (218, 219);
therefore, a detailed discussion will not be included in this article.
Assay control considerations. All NAATs, whether laboratory-developed tests (LDTs)

or FDA-cleared assays, should include a set of controls, including external positive and
negative controls for respiratory viral targets that are tested by all steps in the assay. An
internal amplification control should be added to all specimens except in assays where
inhibition rates of the NAAT have been shown to be below acceptable limits (often
defined by the laboratorian) (220, 221). These controls ensure that target nucleic acid
is recovered and any potential NAAT inhibitors are removed during the sample
processing stage. While commercial NAAT kits are designed to flag invalid results (when
internal controls fail), LDTs require manual checks and result review to detect invalid
specimens. The number of external controls and their frequency of use should be
established by the laboratory based on regulatory requirements and its individualized
quality control plan (IQCP), with a focus on risk assessment. Rules for review and
result-based actions items should be addressed in the laboratory IQCP (222, 223).
Contamination. Molecular target amplification assays are susceptible to false-
positive results caused by contamination, and false-positive results may occur at any
step in sample collection and processing. Preanalytical contamination may occur when
specimen integrity is breached during the collection process or when integrity is
breached during early handling processes in the laboratory (221). Even when using a
biosafety cabinet, steps should be taken to limit the production of aerosols and to
process specimens in a manner that prevents cross contamination (224). Given that
respiratory viruses can be identified in health care environments, it is possible that
inappropriately handled swabs or other specimens could be contaminated with these
viruses (225). It is also possible that a laboratory worker infected with a respiratory virus
may act as a contaminating vector in the laboratory. The greatest risk of contamination
is from amplicons created (and possibly aerosolized) during previous molecular runs.
Most commercial assays using either real-time reporters or array-based detection are
designed to minimize risks of amplicon contamination unless the laboratorian fails to
correctly handle the reaction vessels (221).
Assays that incorporate manual postamplification processing present the highest
risk of contamination to the laboratory. Multiple amplicon sterilization processes have
been established to decrease the chance of amplicon carryover in molecular assays.
These include the use of UV light to create thymidine dimers (cross-linking contami-
nating DNA), altered amplification chemistry using modified nucleotides, addition of
uracil DNA glycosylase (UNG), and the use of hydroxylamine to prevent cytosine and
guanine base pairings in subsequent reactions; however, numerous chemical ap-
proaches may be used (226–228).
Good laboratory practices can also be used to control contamination or carryover of
amplicons (Table 5). These are particularly relevant when multiple processes such as
reagent preparation, nucleic acid extraction, amplification, and postamplification pro-
cessing are utilized. Open systems (where extraction, amplification, and/or detection
stages are exposed to the environment) and closed systems (where extraction, ampli-
fication, and detection are completed within a single compartment not exposed to the
environment) have different contamination control requirements (Table 4). Staff train-
ing protocols and laboratory standard operating procedures (SOPs) should emphasize

TABLE 5 Good laboratory practices for molecular assays

Recommendation for type
of molecular systema
Laboratory practice to decrease contamination events Open Closed
Unidirectional flow (clean to dirty) Recommended Not required
Physical separation of pre- and postwork areas Recommended Not required
Regular decontamination of work areas Recommended Recommended
Use of aerosol-resistant pipette tips Recommended Recommended
Change of PPE between processing steps Recommended Not required
Restricting worker movements postamplification Recommended Not required
Centrifuging reagents Recommended Recommended
Ensuring that only one specimen is uncapped at a time Recommended Recommended
Process to monitor contamination events Recommended Recommended
Dedicated equipment for pre- and postamplification areas Recommended Not required
Monitoring environment for contamination (e.g., by environmental swipe tests) Recommended Recommended

aBased on the type of molecular system, laboratory practices to decrease contamination are either recommended or not required (217, 221–223, 228).
the organization of workflow process (such as unidirectional flow, separate areas for
pre- and postamplification processing, regular decontamination of work areas with
bleach, strict adherence to use of aerosol resistant pipette tips, mandating changing of
gloves and lab coats between processing steps, and restricting work on new samples
after handling postamplification reaction mixtures [228]) and technical practices (such
as aliquoting of reagents, centrifuging of reagents, and care in capping and uncapping
tubes, which may also prevent cross contamination). Physical separation of workspaces
dedicated to different assay steps (e.g., pre-PCR and post-PCR) can also decrease the
risk of contamination (221) and is recommended for open systems, but it is not
necessary for closed systems.
Additionally, laboratorians should develop processes to monitor contamination
events. Sentinel systems, such as running negative or no-template controls in each
amplification assay, can be used for detecting large-scale contamination (221), while
low-level contamination events may be identified by laboratorians as an excessive or
unusual amount of low-level positive specimens (e.g., positive results near the cutoff).
Care should be taken when interpreting results for higher numbers of low-level positive
results outside the normal respiratory virus season, as many low-level positive results
may represent contamination. Care should be taken when interpreting specimens that
are positive for multiple targets, and laboratorians should have a sense of the coinfec-
tion rates within their settings. Coinfection rates may vary widely between adult and
pediatric patient populations and may account for over 10% of all specimens in some
pediatric populations (229–231). Environmental swipe tests should be considered to
monitor workspaces for contamination from current or recently circulating viruses as
well as control materials, and they can be used to detect widespread amplicon
contamination events (232); however, sporadic contamination events may be missed
due to sampling bias. Some FDA-cleared assays have specific recommendations for
environmental monitoring and outline routine decontamination measures. For other
tests, it is up to the laboratory to define intervals as part of their quality assurance
program or IQCP (217, 221–223).
Positive predictive value and false-positive tests. In general, molecular tests for
respiratory viruses have high sensitivity and excellent negative predictive values, which
can reliably rule out infection when assay results are negative. Most molecular assays
for respiratory viruses also have excellent positive predictive values, in the range of 90%
or higher. Because molecular amplification assays for these pathogens are generally
more sensitive than culture-based methods (233), it is often difficult to determine if a
molecular result is a false positive when the reference culture method is negative. In
some instances, a second molecular assay using a different gene target may be used to
resolve discrepant results; however, it should have analytical sensitivity equal to (or
better than) that of the first assay (220). Additionally, when the respiratory viral
pathogen is present at a level close to the assay’s limit of detection, discrepant results

due to Gaussian distribution effects can be observed (234). Finally, sampling error can
affect the results of comparative studies if two separate swabs or collection protocols
are utilized.
Labor and cost of molecular assays. The use of molecular approaches has tradi-
tionally been accompanied by higher supply costs than for antigen- or culture-based
methods (235); however, modern molecular technologies provide improved perfor-
mance characteristics compared to culture and/or DFA/IFA (197). Automation and
integrated molecular test platforms can provide labor savings to the laboratory to offset
increased reagent and platform costs (236) and may also decrease downstream costs
for the health care system by providing more rapid and accurate results. Incorporation
of molecular assays has resulted in variable patient management outcomes depending
on studies, with some studies showing positive effects and other studies showing no
effect, as identified in a recent review by one of the authors of this article (237).

Negative effects on patient management have not been identified. Positive effects on
patient management include decreased patient isolation times (238), length of stay
(LOS) (239), administration of antibiotics and oseltamivir (240), and duration of antibi-
otic therapy (241).
Understanding Applications of Molecular Detection Approaches

Limited role of viral loads in predicting patient outcomes. A growing body of
evidence shows a correlation of respiratory viral load and patient outcome. In one study
of immunocompetent adult patients, age and hospitalization time were associated with
earlier reverse transcriptase PCR (RT-PCR) cycle threshold (CT) values for FLUA/B of ⱕ20
than later CT values (242). Association of viral load and outcome can also vary by
genotype, as RV-A viral loads were higher in patients with severe disease than in
patients without severe disease, while no difference in viral load was observed for
patient groups infected with RV-C (243). Furthermore, increased fatalities in adult CAP
patients were associated with sustained viremia and high viral loads of ADVs in sputum
and tracheal aspirates (244).
However, current laboratory practices generally report qualitative results for a
respiratory virus NAAT, rather than determining a true viral load. The currently available
laboratory-developed viral load assays have multiple problems, including the lack of an
international standard, lack of standardized technology, and lack of consensus on
specimen types (245). Additionally, the timing of specimen collection can influence viral
load results. In fact, viral load samples taken on day 3 postonset may have a stronger
association with clinical outcome than samples taken on day 0, 1, or 2 (246). Given the
viral load data described in this section, the viral shedding data (described above), and
the impact of age, immune status, and/or coinfection with other respiratory viruses
(134), additional studies are needed to determine when viral loads are appropriate in
different patient populations and how to appropriately interpret the results. Due to
sampling errors, time of collection, patient age, etc., viral loads may not be comparable
from one patient to another. In the future, possible roles for these viral load assays may
include monitoring an individual patient over time to assess for viral clearance or
response to antiviral therapy.
Molecular panel testing for respiratory viruses. (i) Defining multiplex assays.
Multiplexing of molecular assays was traditionally restricted by the number of targets
that could be efficiently amplified within a single reaction vessel (247–249). The earliest
approaches were often batch-based assays that relied on a single nucleic acid extrac-
tion followed by one or more molecular assays. Often, panels of multiple individual
targets or small multiplexes with 1 to 3 targets could overcome some of the inefficacies
of massively multiplexed reactions (250); however, development of new technologies
with improved multiplexing capabilities has allowed detection of multiple virus targets
from a single sample (251–253).
(ii) Recommendations for patient populations in which multiplexed respiratory
viral panel testing may be appropriate. Testing requirements may vary depending on
the patient setting and resources, as the costs of the multiplex assays are high. The

most appropriate patients to test may vary depending on the health care setting, as
some studies show questionable utility in testing adult outpatients for viruses other
than FLU (254), Instead, for FLU patients who meet ILI criteria and are at high risk for
complications, a highly accurate rapid test may have the greatest utility. Others have
shown that multiplexed viral panels can directly influence antibiotic utilization practices
(241).
Hematology and oncology patients may be appropriate patient populations for
testing. The Infectious Diseases Working Party of the German Society for Haematology
and Medical Oncology identified community-acquired respiratory virus infection as a
significant cause of morbidity and mortality in oncology patients (103). Infectious viral
etiologies were widely varied and included both single and mixed infections. For
example, RSV infection has a high likelihood of progressing to a lower respiratory tract
infection (30%) and a high chance of mortality (27%) in oncology patients. Therefore,

testing for FLU, RSV, PIV, and other prevalent community-circulating viruses in all
oncology patients presenting with symptoms (103) is suggested.
Transplant patients may also be an appropriate patient population for multiplex
testing. Given the poor predictive value of the U.S. CDC’s ILI criteria not only in adult
transplant patients but in general, some authors have suggested an increased role for
the use of multiplex respiratory NAAT assays in adult transplant patients with suspected
respiratory virus infection (32). In lung transplant patients, identification of mixed viral
infections using a multiplex panel could be used as a predictor of poor outcome (e.g.,
biopsy-proven rejection or sustained decline in forced expiratory volume [FEV1]) (255).
In lung transplant patients, the detection of one or more viruses using a respiratory
virus panel in a BAL fluid sample during the first year after transplant has also been
associated with significantly faster development of bronchiolitis obliterans syndrome
(BOS) (256).
Intensive care unit (ICU) patients may be another appropriate patient population for
respiratory viral multiplex panel testing. In a recent review, respiratory viruses such as
FLU, RSV, and RVs were suggested to cause immunosuppression in ICU patients (257).
Given the clinical severity of illness in patients in the ICU, they are good candidates for
respiratory virus panel testing. Appropriate identification of the severity of patient
illness as well as the patient location (including the ICU) within the health care facility
can often be challenging for the laboratory. Therefore, identification of critically ill
patients with suspected pneumonia has previously been used as a selection criterion in
the absence of accurate hospital location data (258).
Pediatric patients with an underlying illness may also be an appropriate patient
population for respiratory viral panel testing. Panel testing may allow for identification
of pathogens associated with specific risks in pediatric patients. This may include
increased risks for asthma and wheezing in critically ill patients (259) or a lack of FEV1
improvement in pediatric cystic fibrosis patients (260).
(iii) To multiplex or not to multiplex? A variety of commercial and FDA-cleared in
vitro diagnostic tools are currently available. Incorporation of these highly multiplexed
assays into the laboratory significantly decreases turnaround time compared to that
when performing all assays individually (252). Additionally, ease of use is improved with
many assays giving “sample-to-answer” detection of respiratory viruses. Multiplex
assays often have excellent performance characteristics, allowing clinicians to be
confident with test results and make informed clinical decisions with concrete patient
and health system benefits. Compared to complex algorithms involving multiple
ordering of tests for small numbers of viral targets (e.g., FLU, FLU/RSV, EV, and RV
alone), multiplex panels used as a routine test ordering choice can remove some of the
confusion or indecision described by clinicians when ordering tests for smaller numbers
of viral targets individually (261, 262). However, given that these panels are expensive,
demonstration that the results impact patient care help justify the increased cost to the
laboratory. A variety of studies have looked at indirect benefits of multiplexed panel
testing; however, the identified outcomes are not consistent between studies. In
patients 3 months to 21 years old, panel use has been associated with decreased length

of stay (LOS) in emergency departments and inpatient wards (241). Identification of a

viral etiology has also shown improvements in hospital isolation resource use, which
can be removed as appropriate and targeted only to patients who require isolation.
Compared to other methods, multiplex panels can decrease the amount of antibiotic
and antiviral use, and they may be used to appropriately triage patients in acute care
settings (239, 263, 264). A significant decrease in the duration of antibiotic use and the
number of chest radiographs was observed in an adult tertiary care center when rapid
multiplexed panels were used compared to traditional antigen detection and older
molecular methods (239). Adult outpatient outcomes were assessed at a Connecticut
VA Center that used an on-demand respiratory panel. Outpatients were divided into
those with FLU detected, those with a non-FLU virus detected, and those with no
pathogen detected. Antibacterial prescription rates did not vary between groups;
however, there was a statistically significant difference between antiviral prescription

rates: the FLU-positive group was more likely to be treated with an antiviral agent
(80/105 [81%] treated) than were patients in the non-influenza virus pathogen group
(6/109 [5.5%]) and the no-pathogen-detected group (2/81 [2.5%]) (P ⬍ 0.001) (254).
Respiratory panel use allows for more comprehensive characterization of viruses for
general epidemiology/surveillance (15, 265) and outbreak investigation. Other, less
tangible but important, benefits to respiratory viral panel use may also include im-
proved patient and physician satisfaction with an improved test turnaround time.
Multiplexed respiratory virus panels may have significant costs for implementation,
and some may have significant costs to operate. Health care administrators need to be
made aware of the indirect and direct benefits of panels and how cost savings may be
generated through improved workflow practices and lower labor costs (266, 267).
Laboratorians and clinicians may need to reassess how clinical utility studies are
undertaken and consider group efforts to undertake well-controlled and standardized
studies (264).
(a) Multiplexing and the utility of identifying mixed infections. Multiplexing of molecular
assays can facilitate identification of mixed viral infections (268–270). Coinfections are
defined as the detection of more than one virus in a patient specimen. The rate of
coinfection will depend on the particular virus, the methodology used for detection,
the patient population demographics, and the geographic location of the study (271).
However, understanding the impact of coinfections on patient outcomes is challenging,
particularly when molecular tools are used for diagnostics. Nonviable virus from a
remote infection or virus not associated with the current infection may be detected by
molecular methods. Important considerations include (i) whether identification of
mixed infections leads to a better understanding of patient prognosis, (ii) whether
identification of mixed infections leads to changes in patient management, (iii) whether
identification of mixed infections leads to changes in infection prevention and control
practices, and (iv) whether the increased identification of viruses not routinely identi-
fied in nonmultiplex panels allow for placing patients in cohorts based on etiology
during isolation.
In some cases, coinfections may make up a significant proportion of total viral cases
within a population. In one recent study, coinfections with bacteria and viruses were
identified in 40% of viral respiratory tract infections requiring hospitalization (272). For
example, in Japan, a recent study found that 43.8% of patients who were diagnosed
with a CoV infection were also infected with an additional virus (273). In another study,
coinfections of two or more viruses were identified in approximately 18% of infants
with an acute respiratory illness; RV was the most common coinfecting virus, but other
viruses, such ADV, hMPV, and PIVs, were also codetected (270). Thus, the impact of
mixed infection on patient outcomes is still under debate. Some studies show no
difference in patient outcomes when coinfections are compared to single virus-
infections, even in highly immunocompromised patient populations (268). Additionally,
studies in immunocompetent children with lower respiratory tract infection found that
RSV coinfection with any other respiratory virus was not associated with more severe
disease than RSV infection alone (274). Conversely, other studies show that coinfection

with RSV and a second virus in infants with lower respiratory tract infections is
associated with increased length of stay (LOS) (275). In another study, an increased risk
of life-threatening disease (e.g., intensive care unit [ICU] admission, need for mechanical
ventilation, or death) was identified in patients with ADV-RSV coinfections compared to
RSV single-virus infections. In a secondary outcome analysis, FLU-RSV coinfections had
an increase in LOS compared to RSV single-virus infections (274), while ADV coinfec-
tions were more likely to be associated with the need to treat with supplemental
oxygen than were ADV single-virus infections (276). Furthermore, in cases of
community-acquired pneumonia, viral-bacterial infection has been associated with a
more complicated course (e.g., hospital death or mechanical ventilation for ⬎7 days)
than infections with bacteria alone, viruses alone, or no identified etiology (277).
(b) Commercially available molecular test panels may not fulfill all testing needs. A major
drawback of multiplexed panels is the inability to differentiate closely related viruses or

to detect all targets with equivalent sensitivity, and some targets on commercial
multiplex panels continue to be detected more efficiently by singleplex assays (278)
(also see comments on emerging pathogens below). In one study, detection of RSVA
and FLUA had decreased sensitivity in panel tests compared to that with singleplex
NAAT (279). Likewise, detection of ADV in multiplex panels often has decreased
sensitivity compared to that with in-house NAAT assays (280), particularly for ADV
group E (279). Of note, only respiratory species of ADV (B, C, and E) will be detected in
multiplex panels, while nonrespiratory ADV species (A, D, and F) will be missed. In
commercial panels, the proprietary nature of primers and probes does not allow
investigation for detection of emerging viral pathogens, which may be missed by
commercial assays (281).
Another limitation in some available assays is the inability to distinguish EVs from
RVs. This can lead to secondary laboratory differentiation algorithms to characterize
infection (282), and this is compounded by the limited ability to detect emerging EV
strains (278). For example, enterovirus D68 may require altered patient management
compared to seasonal EV strains, as it is associated with extrapulmonary syndromes
such as acute flaccid paralysis (282). Additionally, detection of nonrespiratory ADV in
the respiratory tract can precede systemic infection in immunocompromised children
(283). Unfortunately, there is currently no practical gold standard to determine whether
ADV detection in the respiratory tract is causal or incidental (284).
(iv) Near-patient or POC tests. As highlighted above, CLIA-waived tests are in-
tended for use in “professional” settings (e.g., physicians’ offices, mobile clinics, and
pharmacies) and/or by untrained operators with no laboratory expertise (200). A
summary of NAAT assays that can be used as point-of-care (POC) or near-point-of-care
tests is in Table 4. Technologies for these guidelines are discussed in general here;
specific products are not discussed, and company names are not mentioned.
The availability of newly developed CLIA-waived NAAT assays which detect FLUA/B
or both FLUA/B and RSV is increasing. Multiple assays are now emerging in the
marketplace and may have similar test characteristics (285); users should consult
up-to-date resources for a list of waived products. Users should note that in general,
reverse transcriptase PCR technologies may have higher sensitivities than isothermal
assays (286–289).
Benefits of near-patient NAAT assays include ease of use and reduced process steps
compared to those with older molecular assays, software that allows for easier result
interpretation, and closed systems to reduce contamination (286–289). Drawbacks of
near-patient NAAT assays include the potential to cause unforeseen strain on the
laboratory (e.g., for confirmatory testing and quality assurance program support), the
impact on resource utilization outside central laboratories, and the limited scope of
specimen types that can be used (290–292).
A recent review of POC testing, including NAAT, identifies several barriers to
understanding the benefits of point-of-care testing for respiratory viruses (237). Imple-
mentation of rapid nucleic acid testing could be associated with decreases in number
of hospital admissions, length of stay, emergency department length of stay, duration

of antimicrobial use, droplet contact days, total isolation days, and receipt of antibiotics
(238–241).
Appropriate Test Utilization in the Era of Molecular Testing

Respiratory virus testing algorithms vary between health care institutions. Re-
sources, types of laboratory facilities, and different patient populations (to name a
few) may all play a role in the testing algorithm chosen. Choosing Wisely is a
campaign started in 2012 that focuses on initiating discussions with both the
patient and physicians about unnecessary procedures, treatments, and tests (293).
This section focuses on Choosing Wisely and discusses (i) which testing options
might be suitable to perform depending on needs, (ii) what laboratories can do
when resources are limited, (iii) how the importance of preanalytics plays into the
testing decision being made, and (iv) what additional considerations need to be

discussed up front before any test or piece of equipment is adopted by the
laboratory or health care environment. The following sections describe key steps in
ensuring that health care workers choose respiratory tests appropriately.
Stakeholder engagement. To provide high-quality, cost-effective laboratory ser-
vices, it is imperative to understand the clinical needs of the end users when consid-
ering solutions for detection of respiratory viruses (294). Depending on the health care
system, the laboratory may be asked to offer testing within the main laboratory or to
play a role in determining the best test for near-patient testing. Because diagnostic
needs vary, it is important to identify the right stakeholders at the beginning in order
to determine appropriate process development and assay deployment.
Stakeholder discussion should include the needs of primary care providers, charac-
teristics of the patient population, clinical practice settings, required test turnaround
time, availability and expertise of nonlaboratory staff to perform POC testing, the
volume of testing, and potential outcomes of a new assay/process. Physician groups
utilizing testing are broad and may include the emergency department, inpatient/ICU,
infection prevention and control groups, and pediatric and adult outpatient services
such as urgent care or family practice. The laboratory, along with infectious diseases
physicians, should engage these providers to completely understand the provider/
patient need.
In order to choose wisely for respiratory virus testing, one must have a fundamental
understanding of the needs of the organization. Early engagement with the provider
and operational stakeholders (departmental administrators or managers overseeing
specimen collection and/or testing) is paramount to successful test implementation. It
is crucial for an institution to consider and understand the potential clinical and
financial impact of a diagnostic test. Some decisions may be made based on outcome
data in the literature or data that are internally generated (263, 295–304). Outcomes
can include (but are not limited to) cost, TAT, infection prevention and control
decisions, antibiotic administration, antiviral administration, inpatient LOS, rates of
admission to the hospital, referrals, and ancillary testing (chest radiography or other
laboratory testing) (299, 302). A positive or defined outcome not only demonstrates the
utility of a specific test but can also be presented to administrators to support the
proposal. Many institutions today are implementing test algorithm changes in part due
to evidence-based medicine and outcome data.
A PubMed search for the terms “respiratory,” “virus,” “testing,” “utilization,” and
“compliance” found no articles related to utilization and compliance for respiratory
virus testing; however, we have identified a need for monitoring usage after imple-
menting algorithms to ensure compliance and appropriate utilization of tests by the
ordering health care workers.
Choosing the right test. As evidenced by the diversity of institutional provider
groups discussed above, a single solution might not work for all patient populations
or specialties of care. In choosing wisely, regardless of the test or the ability to be
reimbursed, the emphasis should be on what the provider will do with the result
and how implementation will impact the clinical outcome, the quality of care given

to the patient (e.g., reduction in unnecessary antibiotic use or duration) or the

institution (e.g., reduced length of stay [LOS] in the hospital). Because many
laboratories are being asked to do more with less, it is incumbent on not just the
laboratory personnel, but all health care professionals, to spend money wisely and
show the impact of testing that is implemented. Quality of care is also improved
when physicians understand how to best use a result from a laboratory test. In
many electronic medical records (EMR), decision trees can be adopted to aid in
appropriate test selection, and tests can be restricted by patient location (e.g.,
inpatient versus outpatient) to promote effective ordering habits. As fee-for-service
models are replaced with integrated care delivery systems, test reimbursement
becomes less of a driver for best practices for respiratory virus testing. For example,
laboratorians should consider the importance of providing influenza A virus sub-
type data when using/considering molecular assays, as some FDA-cleared tests do

not provide the subtype. In some settings, clinicians may not voice concerns about
lacking subtype information. An argument against subtyping is that subtyping
matters only when circulating subtypes have different patterns of resistance to
antiflu drugs. In other settings, clinicians may use subtyping data to place patients
in cohorts in health care settings with low bed-to-patient ratios.
As described above, many providers have historically relied on RADTs, culture, or
direct fluorescent-antibody (DFA) testing for the detection of respiratory viruses. RADTs
have still maintained their popularity because of their rapidity even though they are
suboptimal in regard to sensitivity (209, 305). Over the last decade, the use of NAATs
with relatively faster sample-to-answer times has replaced that of more traditional
methods (306). Sample-to-answer methods with TATs of ⬃1 h may be acceptable for
hospitalized patients, or perhaps patients in the ED, but TATs exceed those required in
outpatient setting. More recently, FDA-cleared and CLIA-waived NAATs with sensitivi-
ties and specificities comparable to those of FDA-cleared laboratory-based molecular
tests have become available (307).
Complex multiplex PCR assays are often restricted to hospital settings and
reserved for the most ill patients with associated comorbidities. Diagnosis of
respiratory illness in this setting is deemed important to the physician even though
treatment might not be available for a specific pathogen. The infection prevention
and control needs of a health care institution may warrant the implementation of
multiplexed testing to appropriately place patients with similar infections in cohorts
when bed space is restricted. These multiplex assays can be further divided into
random-access and batched testing platforms (306). Both routine and unplanned-
for laboratory needs may require the laboratorian to consider utilizing both
batched testing and random-access test systems. Random-access platforms are
suggested for daily use in laboratories with low to medium specimen volumes, with
the benefit of a rapid turnaround time and simplified workflow. As test volumes
increase, the laboratorian may reconsider test algorithms and utilize a batched
testing platform (308). Some algorithms may improve cost-effectiveness by offering
a less-expensive upfront singleplex assay for FLU or duplexed or triplexed assays,
including FLU and RSV, and using multiplex panels only if the sample is negative for
FLU; however, algorithms will vary by institution, time of year, and prevalence of
influenza. Furthermore, algorithms should be chosen based on stakeholder engage-
ment and the individual testing needs of the patient population.
So, how is this made operational? We have provided a risk assessment flow chart in
Fig. 2. We realize that a single approach will not be applicable to all laboratories.
Therefore, laboratorians should work with their clinical partners and manufacturers to
establish risk-based algorithms which can be used to determine the appropriateness of
testing. Test ordering systems, clinical information, and patient location, as well as
demographic identifiers, can be used to streamline the placing of specimens into
appropriate test algorithms (e.g., no testing, testing for limited targets, or broad panel
testing). Laboratorians should offer clinicians the opportunity to discuss cases that do


FIG 2 A risk assessment approach to determine populations most effectively served by acute respiratory virus
testing. The decision-making model can be used to identify the level of test complexity for patient
populations.
not fit into general risk groups (e.g., low versus high), where patients may benefit from
specific laboratory tests.
Recent Issues Surrounding LDTs for the Diagnosis of Acute Respiratory Viral
Infections
LDTs may find a role in the clinical laboratory under the following scenarios: where
commercial assays are not available, when performance issues emerge with commercial
respiratory virus assays, or when a new assay is required immediately (e.g., in the event
of an emerging respiratory pathogen) (309). LDTs are defined as assays developed and
performed by high-complexity laboratories (e.g., “home brew” or “in-house” assays)
that are “intended for clinical use” (310). Draft guidance documents surrounding LDT
use were released in 2014 by the FDA, which provide guidance for clinical laboratories,
industry, and drug administration staff (310). As of 2016 to 2017, the FDA proposed a
“risk-based, phased-in approach, in combination with continued exercise of enforce-
ment discretion for certain regulatory requirement and certain types of LDTs”; however,
it is up to the individual laboratory to calculate the risk associated with the use of LDTs
(311). These issues are not specific to the United States (312). This proposed framework

would place each LDT into a specific risk class (305), and laboratories in other countries
may benefit from comparing how they and their U.S. colleagues perform risk assess-
ments (313).

Older methods such as rapid antigen detection techniques, DFA tests, and viral
culture have essentially been replaced by more rapid and sensitive NAAT assays, which
have improved the characteristics of laboratory tests for the diagnosis of acute respi-
ratory viral infections. However, the highly sensitive nature of these tests as well as the
possibility for molecular contamination means that laboratorians need to develop
processes and practices to prevent molecular contamination. Laboratorians should
understand the risks and benefits of using LDTs and potential regulations restricting
their use. Rapid POC NAATs are allowing for the rapid detection of multiple respiratory

pathogens compared to routine laboratory NAATs. Multiplexed NAATs, including POC
tests, now allow for rapid detection with faster turnaround times (TATs) and sensitivity
and specificity equivalent to those of laboratory-based NAATs. Apart from patient
management for FLU, the patient and system benefits of multiplexed NAATs require
further study, and current study outcomes may be confounded by multiple factors.
Laboratorians should consider strong utilization approaches when initiating supporting
NAAT POC test and multiplexed NAAT implementation. Laboratory utilization discus-
sions should take into account the clinical utility of testing in specific patient popula-
tions. Finally, although NAATs are the primary method of detection, laboratorians
should coordinate testing in a reference laboratory that undertakes viral culture
techniques to allow for phenotypic influenza virus characterization and/or antiviral
susceptibility testing as part of ongoing public health surveillance.
ANTIVIRAL AND PROPHYLACTIC AGENTS: IMPACT ON THE CLINICAL

LABORATORY
RSV Prophylaxis and Antiviral Agents
The use of palivizumab (314) has been described above. Laboratory diagnosis of RSV
has no direct impact on the decision-making on when to initiate palivizumab prophy-
laxis, but general laboratory testing trends may help in the determination of when the
RSV season starts and ends in some locations (95).
Although the use of multiple agents to treat respiratory viral infections has been
described, the number of antiviral agents with FDA approval is limited. For treatment
of RSV infection, the only approved agent is ribavirin (in aerosolized form). The use of
aerosolized ribavirin can pose health hazards to health care workers and is not easy to
deliver to patients, making it a less-than-ideal treatment choice. The 2012 Report of the
Committee on Infectious Diseases (Red Book) focuses on pediatric infections and indi-
cates that primary treatment for RSV is supportive. The Red Book does not recommend
the routine use of ribavirin but does indicate that use may be considered in “selected
patients with documented, potentially life-threatening RSV infections” (315). Research-
focused approaches regarding RSV mutations is not described further here; however,
potential mutations driving resistance against palivizumab and issues with ribavirin are
described in a recent review (215). A comprehensive review of the effectiveness of
antivirals for these viruses is beyond the scope of this guidance document, but there
are emerging data supporting the use of oral ribavirin in treatment of URTI and LRTI in
stem cell transplant patients (102, 103, 316–318). As new antiviral agents for RSV (and
other viruses) become approved, laboratorians may need to develop processes for
systematic antiviral resistance testing and surveillance.
Treatment and Prevention of Influenza

FLU is the only respiratory virus discussed in these guidelines that currently has a
vaccine available for prevention (315). Clinical laboratories should work with their
public health laboratories to ensure that appropriate FLU characterization by culture
and molecular methods occurs. Culture may still be required for phenotypic strain

typing as well as antiviral susceptibility testing as part of studies or national surveillance

systems. These data may also help support decision-making regarding FLU vaccine
effectiveness (41).
Currently licensed antivirals for influenza include the adamantanes, which block
the activity of the M2 protein (active only against FLUA), and neuraminidase (NA)
inhibitors (NAIs), which block the activity of the NAs of influenza A and B viruses. At
the time of this publication, NAIs are the only drugs that are effective for the
prevention or treatment for influenza. Adamantanes, which do not have activity
against FLUBs, are no longer effective against seasonal FLUA (319). Two NA inhib-
itors, oral oseltamivir and inhaled zanamivir, are licensed in many countries. In
addition, intravenous peramivir is licensed in Japan, China, South Korea, Canada,
and the United States. A fourth drug of this class, long-acting inhaled laninamivir,
is licensed in Japan. Similar to the case for M2 blocker-resistant viruses, viruses

resistant to an NA inhibitor(s) may gain an evolutionary advantage and spread
beyond countries employing NA inhibitor therapy. In 2007 to 2009, oseltamivir-
resistant A(H1N1) seasonal prepandemic viruses rapidly emerged and spread glob-
ally (320, 321). In contrast, influenza A H1N1 (pdm09) virus strains are almost
universally susceptibility to oseltamivir and zanamivir (322). Continuous antiviral
susceptibility testing of seasonal FLU viruses is imperative to identify and track the
emergence and spread of viruses resistant to NA inhibitors and M2 blockers.
Relevance of FLU Antiviral Resistance Testing

Guidelines from the Community Network of Reference Laboratories for Human
Influenza in Europe suggest that testing for antiviral resistance is typically indicated in
the following instances: (i) in patients lacking virological improvement (persistent virus
shedding after 5 days of treatment using ab NAAT that “delivers semi-quantitative
information” [e.g., a real-time PCR with a CT value]), (ii) in patients treated with antivirals
with severe FLU who do not clinically improve (time frame not given), (iii) in fatal cases
where an understanding of resistance may influence prophylaxis of contacts, (iv) in
cases of FLU developed during or after antiviral prophylaxis, and (v) in contacts of
antiviral-treated FLU patients who developed respiratory symptoms or in contacts of
FLU patients for whom the presence of resistant virus had been confirmed (323). One
group that may benefit from antiviral testing is patients who shed virus for long periods
of time and who do not improve after treatment (e.g., highly immunocompromised
patients) (324, 325).
As molecular markers of resistance are not well established and vary depending on
virus type/subtype and NA inhibitor, determination of antiviral resistance should be
carried out in a reference laboratory with experience in these techniques (326). Doc-
uments created by the WHO’s Global Influenza Surveillance and Response System
(GISRS) and the WHO Influenza Antiviral Working Group (WHO-AVWG) can assist in the
interpretation of these results (327, 328). Other documents may be available from other
committees which provide guidance on the use of influenza antivirals (329).

Laboratorians should identify a reference laboratory for the characterization of
influenza and antiviral susceptibility testing. Antiviral testing is not a routine test, and
the time required to undertake such testing limits the clinical relevance of this testing
in most patient populations. Antiviral testing may be required for epidemiological
studies as well as cases of failure in prophylaxis. One patient population that may
benefit from this testing is patients who are highly immunocompromised who do not
clinically improve following antiviral treatment and who may shed virus for an ex-
tended period of time.
CODING AND REIMBURSEMENT

This section was introduced into the guidance document following presentation of
these guidelines in the draft from at an ASM general meeting. Current procedural

terminology (CPT) is a set of guidelines, codes, and descriptions used to elucidate and
standardize services by health care professionals, including testing in the clinical
laboratory. The CPT codes for microbiology and virology are established through the
Pathology Coding Caucus (PCC) of the American Medical Association (AMA). CPT codes
in microbiology and virology have a 5-digit identifier with a description of the target
and procedure (e.g., 87,633, CPT code in the category “infectious agent detection by
nucleic acid [DNA or RNA]”). New codes are published yearly. Inclusion of a code in the
CPT manual does not imply endorsement of the test, nor does it cover insurance or
reimbursement policies.
In general, when a new test that needs a code is available, a proposal for coding is
presented to the PCC. Among the criteria used by the PCC to review the request are test
methodology definition, the volume of test utilization, the medical necessity, and
scientific publications detailing performance and outcomes studies for the new test.

After each caucus meeting, a document entitled “CPT Editorial Summary of Panel
Actions” is prepared, which summarizes the actions that were taken by the panel on
each of the code applications.
Pricing/fee setting for a CPT code is the purview of the Centers for Medicare and
Medicaid Services (CMS). Annually, the CMS holds the Clinical Laboratory Fee Schedule
(CLFS) meeting at its headquarters in Baltimore, MD. Stakeholders present the code(s)
(as established by the PCC) and a proposed reimbursement amount (based on an
existing rate or as a recommend new rate based on a comprehensive cost analysis). The
CMS Advisory Panel on Clinical Laboratory Diagnostic Tests functions to establish
payment rates based on crosswalking or gapfilling and establishes factors used for
determining coverage and payment processes (330).
Per the CMS (331), crosswalking occurs when a new test (or substantially revised
test) is determined to be similar to multiple existing test codes, portions of an existing
test code, or an existing test code. Gapfilling occurs when there is no existing compa-
rable test available (331).
As of 2017, reimbursement compliance is a system in place to ensure that the
testing being performed is medically relevant for the clinical situation. Here, appropri-
ate testing for specific clinical conditions and clinical outcomes is critical. The issue of
medical relevance has been raised in virology recently in regard to multiplex respiratory
virus and gastrointestinal panels. In brief, CPT code 87,633, defined as respiratory virus
(e.g., ADV, FLU, CoV, hMPV, PIV, or RV), includes multiple NAAT reactions, and multiplex
NAAT panels with target numbers (including types or subtypes) ranging from 12 to 25
targets. The medical necessity and reimbursement for these multiplex assays have been
challenged, and Medicare and Medicare administrative contractor (MACs) alerted pro-
viders that a “broad-net” or “one-size-fits-all” panel contributes to test overutilization
and increased health care costs without specific benefit to a given patient. They assert
that testing should be limited to organisms with the greatest likelihood of occurrence
in a given patient population and, if results are negative, to provide reflexive testing to
more “exotic” organisms.
A consortium of clinical organizations whose members represent testing laborato-
ries has submitted comments directly to MACs, recommending a thorough review of
this issue. At the time of this writing, only a partial resolution has occurred (as per verbal
communication by one of the authors).
Payment rates continue to be under scrutiny and have been discussed during
implementation of the Protecting Access to Medicare Act (PAMA). This statute calls
for a market-based fee schedule based on a weighted median of individual private
payor test reimbursements reported by “applicable laboratories,” which by specific
requirements excluded hospital laboratories. Applicable laboratories included 45%
of all commercial laboratories and 5% of physician office laboratories. As such, the
data for reimbursement are heavily weighted by discounted pricing by large
commercial entities to major payors (MACs). Beginning in January 2018, the inten-
tion was for price reductions to be implemented at 10% in each of the next 3 years,
followed by a 15% reduction for the following 3 years, until the established

weighted median price is hit. These new fees were to be applied to all who are paid
using the CLFS. Of note, concerned organizations and individuals have contacted
CMS about the detrimental effect of the act and the predicted closure of many
laboratories and the impact on patient care. The status of these new fees was in
question as of January 2018 (332).

Laboratorians should be aware of reimbursements for existing and new diagnostics
for respiratory in their locations.
CONCLUSIONS
This is the most recent update of ASM practice guidelines for clinical microbi-
ology, addressing changes to acute respiratory viral infection diagnostics since the

previous document, which was published in 1986. Since that time, laboratory
practices as well as clinical practices have changed extensively. The guidelines were
developed for the laboratory diagnosis of viruses causing acute respiratory illness,
with technologies ranging from low- to high-complexity testing. Respiratory virus
testing may be considered if a diagnosis has impact on patient management,
especially when FLU treatment decisions are based on test results or in immuno-
compromised patients. In general, testing immunocompetent patients will not
impact patient management. However, testing may be undertaken for surveillance
in sentinel labs, to guide infection control decisions/practices, or when highly
pathogenic emerging pathogens are suspected.
The landscape of respiratory virus testing has significantly changed in the last
30 years. The decreased use of older technologies such as viral culture and direct
antigen detection represents a significant programmatic change in the diagnosis of
respiratory viruses. Many front-line clinical laboratories have completely phased out
viral culture, and testing such as strain typing and antiviral resistance testing is
generally limited to reference laboratories. Molecular techniques are now the preferred
diagnostic approaches for the detection of acute respiratory viruses and are more
amenable to automation and high-throughput workflows. Good molecular laboratory
practices and quality assurance programs are keys to preventing laboratory contami-
nation. The decreasing complexity of platforms used for molecular testing has ex-
panded the geographic capacity of these assays, which can now be placed closer to
patients as POC tests, while newer technologies have made multitarget panels widely
available. For novel and emerging respiratory viruses, laboratory-developed tests will
still be required to compensate for testing gaps that often need to be filled quickly.
With all the advances in technology, however, effective communication between
clinicians and the laboratory is still essential to quickly identify highly transmissible
emerging pathogens and reduce health care worker exposure. Laboratorians should
work closely within their teams as well as with other clinicians and public health
practitioners to ensure that health systems are prepared for the inevitable emergence
of new respiratory viral pathogens.
Implementation of clinically relevant testing algorithms can ensure optimized patient
care and improve laboratory resource management. Particularly, strong preanalytical
screening approaches can facilitate appropriate specimen collection and direct providers to
correctly order diagnostic tests as needed. Laboratorians should ensure that they continue
to work with their public health reference laboratory colleagues to align processes to
enable continued virus characterization and antiviral resistance testing.
ACKNOWLEDGMENTS
We thank Tatiana Baranovich (Carter Consulting, Inc., Influenza Division, National
Center for Immunization and Respiratory Diseases, Centers for Disease Control and
Prevention, Atlanta GA, USA) and Larisa V. Gubareva (Influenza Division, National Center
for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention,
Atlanta GA, USA) for contributing comments and expert opinion on relevance of FLU

antiviral testing, and we thank Stephen Lindstrom (Diagnostics Development Team,

Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA) for
contributing general comments and expert opinion on FLU diagnostics.
Yi-Wei Tang’s contribution to this work was supported in part by NIH/NCI Cancer
Center Support Grant P30 (CA008748) and in part by research agreements between
the Memorial Sloan-Kettering Cancer Center (MSKCC) and the Luminex Corporation
(SK2013-0929) and between the MSKCC and Cepheid (SK2017-1885).
Carmen L. Charlton and Steven J. Drews conceptualized the original manuscript
design, wrote multiple sections, recruited coauthors, and organized contributions
following input from other authors; they both elicited feedback from ASM members at
the General Meeting. Yvette S. McCarter and Audrey N. Schuetz provided critical
feedback on the original design of the manuscript, the recruitment of authors, and the
scope of the document and provided general writing comments during feedback

phases. Yvette S. McCarter also provided feedback during the working session at the
ASM General Meeting. Esther Babady and Yi-Wei Tang wrote sections on the epidemi-
ology and clinical presentation of respiratory viral infections and provided feedback on
the larger combined document. Yi-Wei Tang presented the concept to and elicited
feedback from ASM members at the General Meeting. Christine C. Ginocchio provided
writing on near-patient testing and presented the concept to and elicited feedback
from ASM members at the General Meeting. Todd F. Hatchette and Mike Loeffelholz
wrote the section on specimen collection and provided general writing feedback on
revisions of the document. Yan Li provided writing on the influenza diagnostics
sections and general writing feedback on revisions of the document. Robert C. Jerris
wrote the section on coding and reimbursement and provided general writing feed-
back to revisions of the document. Susan Novak-Weekley wrote sections on appropriate
test utilization and provided general writing feedback to revisions of the document.
Melissa B. Miller and Ray Widen wrote sections on molecular testing and provided
general writing feedback to revisions of the document.
Esther Babady has received research funding for evaluation of respiratory pan-
els/tests from Luminex Corp and GenMark Diagnostics. Carmen L. Charlton has
received grant funding from Merck Pharmaceuticals. Steven J. Drews is a content
expert involved in assisting Johnston & Johnston (Janssen) Pharmaceuticals on a
literature search for point-of-care testing for respiratory viruses. Christine C. Ginoc-
chio is an employee of bioMérieux and BioFire Diagnostics and owns stock in
bioMérieux. Todd F. Hatchette has grant funding from GSK and Pfizer for
investigator-initiated research (severe outcome surveillance for influenza and CAP
examining influenza and pneumococcal vaccine effectiveness). Melissa B. Miller is a
Scientific Advisory Board member for Abbott Molecular, BioFire Diagnostics, Curetis,
and Luminex Molecular Diagnostics and has received grant or study support from
Cepheid, Curetis, Hologic, and Luminex. Susan Novak-Weekley has recently been on
an advisory board for Roche Molecular and has worked for and received payment
from Copan, Qvella, Gen Mark, and Asolva. Audrey N. Schuetz discloses grant money
from Merck Pharmaceuticals. No conflicts of interest are declared by Mike Loeffel-
holz, Robert C. Jerris, Yvette S. McCarter, Yan Li, and Ray Widen.
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Carmen L. Charlton, Ph.D., M(ASCP)CM, Christine C. Ginocchio, Ph.D., MT(ASCP), is
F.C.C.M., D(ABMM), is a clinical microbiolo- Vice President, Medical Affairs, bioMérieux,
gist at the Provincial Laboratory for Public Vice President, Scientific and Medical Affairs,
Health (ProvLab) and Assistant Professor of BioFire Diagnostics, and Professor of Medi-
Laboratory Medicine and Pathology at the cine, Northwell School of Medicine, New
University of Alberta. Her current research York. She has 40 years of experience in all
focuses on vaccine-preventable diseases, phases of laboratory management and clin-
with emphasis on infections passed from ical diagnostics. She has been the principal
mother to infant during pregnancy. She ob- investigator for more than 60 diagnostic in-
tained her Ph.D. in biochemistry and bio- dustry and pharmaceutical clinical trials, in-
medical sciences from McMaster University cluding 23 studies of in vitro diagnostic de-
in 2010 and completed her clinical microbiology training at the Univer- vices for U.S. FDA clearance. Her areas of extramural funded research
sity of California, Los Angeles (UCLA), in 2013. Dr. Charlton is the included HIV, cytomegalovirus, respiratory viruses, human papillomavi-
Virology Program Leader for HIV, Hepatitis, Prenatal and Immunity rus, antibiotic resistance, and molecular diagnostics for infectious dis-
Screening, and Human Papillomavirus at ProvLab. She is incoming eases. Her awards include the President’s Award and Irving Abrahams
President of the Canadian Association for Clinical Microbiology and Award for outstanding basic science research, the PASCV 2012 award in
Infectious Disease (CACMID), served on the American Society for Micro- diagnostic virology, and an ASM 2013 BD award for research in clinical
biology’s (ASM’s) Practical Guidance for Clinical Microbiology Commit- microbiology. She is co-editor-in-chief for the Journal of Clinical Virol-
tee, and is a current member of the Clinical and Public Health Micro- ogy, a section editor for the 10th, 11th, and 12th editions of the Manual
biology planning committee for the ASM Microbe meeting. of Clinical Microbiology, and on the editorial board for Clinical Microbi-
ology Reviews.
Esther Babady, Ph.D., D(ABMM), is the Di-

rector of Clinical Operations for the Microbi- Todd F. Hatchette, M.D., F.R.C.P.C., com-
ology Laboratory Service, Director of the pleted his M.D. and internal medicine resi-
CPEP Clinical Microbiology Fellowship pro- dency at Memorial University of Newfound-
gram, and an associate attending microbiol- land. After completing residency training in
ogist in the Department of Laboratory Med- infectious diseases and medical microbiol-
icine at Memorial Sloan-Kettering Cancer ogy at Dalhousie University, Dr. Hatchette
(MSKCC) in New York, NY. She received her completed postdoctoral research training in
Ph.D. in biochemistry and molecular biology virology at St. Jude Children’s Research Hos-
and completed a postdoctoral CPEP fellow- pital in Memphis, TN. Dr. Hatchette is the
ship in clinical microbiology, both at the Chief of Service for the Division of Microbi-
Mayo Clinic in Rochester, MN, before joining MSKCC. She is board ology, Department of Pathology and Labo-
certified by the American Board of Medical Microbiology and serves on ratory Medicine, in the Nova Scotia Health Authority’s Central Zone and
the editorial boards of the Journal of Clinical Microbiology and Journal of a Professor in the Department of Pathology with cross-appointments in
Molecular Diagnostics. Her research interests include rapid diagnosis of the Departments of Immunology and Microbiology and Medicine,
infections in immunocompromised hosts and the development and where he is a consultant in infectious diseases. Dr. Hatchette has
evaluation of the clinical utility of molecular microbiology assays. expertise in the clinical and laboratory diagnosis of viral infections and
serves as an advisor on a number of local, provincial, and national
committees. He is the President of the Association of Medical Microbi-
ology and Infectious Disease, Canada.
Continued next page

Robert C. Jerris, Ph.D., D(ABMM), is Medical Yvette S. McCarter, Ph.D., D(ABMM), is a Pro-
Director, Microbiology, at Children’s Health- fessor of Pathology and Laboratory Medicine
care of Atlanta and Adjunct Professor at Em- at the University of Florida College of
ory University School of Medicine. He com- Medicine-Jacksonville and Director of the
pleted his Ph.D. in experimental pathology Clinical Microbiology Laboratory at UF
at Emory University in 1981 and a postdoc- Health Jacksonville. Dr. McCarter received
toral fellowship at the Centers for Disease her Ph.D. in pathology from the Medical Col-
Control and Prevention. He has concen- lege of Virginia and completed a postdoc-
trated his research career in rapid methods toral fellowship in medical and public health
(molecular and matrix-assisted laser desorp- microbiology at Hartford Hospital under the
tion ionization–time of flight [MALDI-TOF]) mentorship of Dr. Raymond C. Bartlett and
for microbial identification and antimicrobial resistance. Dr. Jerris is the Dr. Ann Robinson. She is a diplomate of the American Board of Medical
founding father of the South Eastern Association for Clinical Microbiol- Microbiology. Dr. McCarter recently completed a term as Chair of the
ogy, has served for over 30 years in numerous positions with the American Board of Medical Microbiology. She is the microbiology as-
American Society for Microbiology, and currently is the Chair of the sociate editor for Lab Medicine and currently serves on the American

ASM Committee on Professional Affairs. Society for Clinical Pathology LabQ editorial board and Workshops and
On-Demand Webcasts Committee. Her research interests include utili-
zation controls in the clinical microbiology laboratory, cost-effective
Yan Li joined the National Microbiology Lab- laboratory medicine, and evaluation of new diagnostic tests.
oratory (NML) of the Public Health Agency of
Canada in September 1998. He is the Chief of
the Influenza and Respiratory Viruses Section Melissa B. Miller, Ph.D., D(ABMM), F(AAM),
of NML. He received his Ph.D. from the Uni- is a Professor of Pathology and Laboratory
versity of Ottawa in 1992. As the head of the Medicine at the University of North Carolina
WHO National Influenza Center in Canada, at Chapel Hill School of Medicine. She has
Dr. Li takes a leadership role in planning, been the Director of the Clinical Mycobacte-
developing, and maintaining a comprehen- riology, Mycology, and Molecular Microbiol-
sive national surveillance programs for influ- ogy Laboratories and Associate Director of
enza virus and other respiratory pathogens. the Clinical Microbiology-Immunology Labo-
He provides ongoing scientific advice and technical expertise to stake- ratory at UNC Medical Center since 2004. Dr.
holders in influenza surveillance and diagnostics. He has 187 peer- Miller received her Ph.D. in molecular biol-
reviewed publications. ogy from Princeton University and com-
pleted the Medical and Public Health Microbiology Fellowship at UNC.
She has a long-standing interest is respiratory viral diagnosis, including
Mike Loeffelholz earned a Ph.D. in microbi- assessing new technologies, determining appropriate testing algo-
ology from Ohio University in 1987 and com- rithms, and assessing impact on patient outcomes. As a past member
pleted a postdoctoral fellowship in medical and chair of the ASM Committee on Laboratory Practices and current
and public health microbiology at the Uni- chair of the ASM Professional Practice Committee, Dr. Miller has a keen
versity of Rochester in 1990. Dr. Loeffelholz is interest in establishing best practices and advocating for standardiza-
currently a Professor in the Department of tion and dissemination of these practices.
Pathology and Director of the Clinical Micro-
biology Laboratory and the American Soci-
ety for Microbiology CPEP-accredited Medi- Susan Novak-Weekley, Ph.D., S(M)A.S.C.P.,
cal Microbiology Fellowship program at the D(ABMM), is the Vice President of Medical
University of Texas Medical Branch at Galves- Affairs at Qvella. Dr. Novak-Weekley re-
ton. Dr. Loeffelholz has over 80 peer-reviewed publications and book ceived her B.S. in microbiology at Colorado
chapters. He is editor-in-chief of the Clinical Virology Manual, 5th edi- State University and her Ph.D. in microbi-
tion, and an editor of the Journal of Clinical Microbiology. Dr. Loeffelholz ology at the University of Arizona. She com-
has served on a number of committees at the national level, including pleted a postdoctoral fellowship in clinical
the ASM Committee on Professional Affairs, the CDC Board of Scientific microbiology at the University of California,
Counselors/Office of Infectious Diseases, and the Association of Public Los Angeles, Medical Center and Wadsworth
Health Laboratories board of directors. He is a diplomate of the Amer- VA Medical Center in Los Angeles, CA. Dr.
ican Board of Medical Microbiology (ABMM). Novak-Weekley is currently a member of the
American Society for Microbiology (ASM) Journal of Clinical Microbiology
editorial board. Dr. Novak-Weekley is currently the Councilor for the
Southern California American Society for Microbiology (SCASM), in
addition to serving as annual meeting and fundraising cochair. Addi-
tionally, she is a member of the Committee on Microbial Sciences
(COMS) for ASM and also serves on the Nominating Committee for ASM
and the New Technologist Mentoring Committee (CMMC).

Audrey N. Schuetz, M.D., M.P.H., D(ABMM), Ray Widen is the Scientific Director, Esoteric
is an Associate Professor of Laboratory Med- Testing and Research, Tampa General Hospi-
icine and Pathology at the Mayo Clinic tal. He has more than 34 years of clinical
School of Medicine and Science in Rochester, microbiology experience, 28 years of flow
MN. She received her M.D. and completed a cytometry research experience, and over 24
pathology residency and medical microbiol- years of molecular diagnostics assay devel-
ogy fellowship at Emory University School of opment and validation expertise. Dr. Widen
Medicine. She is board certified in clinical has an extensive background in applications
pathology, anatomic pathology, and medical in leukemia/lymphoma diagnostics as well
microbiology through the American Board as infectious disease diagnostics for viral,
of Pathology and is board certified by the bacterial, and fungal targets.
American Board of Medical Microbiology. Dr. Schuetz is Director of
Initial Processing and Media Laboratories and Co-Director of Bacteriol-
ogy in the Division of Clinical Microbiology at Mayo. She is a member Steven J. Drews, Ph.D., F.C.C.M., D(ABMM),
of the Expert Panel of Microbiology of the Clinical and Laboratory is a clinical virologist at the Provincial Labo-

Standards Institute. Her interests include infectious diseases pathology ratory for Public Health (ProvLab), Alberta,
and evaluation of rapid diagnostic techniques to improve antimicrobial Canada. He completed his Ph.D. in experi-
stewardship. mental medicine (infectious diseases) in
2003 at the University of British Columbia.
He then completed his clinical microbiology
Yi-Wei Tang is currently the Chief of the fellowship at the University of Toronto. Since
Clinical Microbiology Service at the Memo- then, he has focused on both the clinical
rial Sloan-Kettering Cancer Center and a Pro- microbiology and research aspects of respi-
fessor of Pathology and Laboratory Medicine ratory infections and has been involved in
at the Weill Medical College of Cornell Uni- respiratory virus surveillance and preparedness planning at a national
versity in New York, NY, USA. He obtained level in Canada. Dr. Drews currently heads the province-wide influenza
his medical training from Fudan University and acute respiratory viral diagnostics program at ProvLab, Alberta. Dr.
Shanghai School of Medicine and Ph.D. in Drews has held faculty positions at both the University of Toronto and
microbiology and immunology from Vander- the University of Calgary. He is currently an Associate Professor in
bilt University. He has been engaged in med- Laboratory Medicine and Pathology at the University of Alberta, Ed-
ical and molecular microbiology transla- monton, Canada, and the outgoing President of the Canadian College
tional research, aimed at developing and evaluating new and advanced of Microbiologists.
microbiological diagnostic testing procedures. Dr. Tang is a Fellow of
the American Academy for Microbiology and of the Infectious Disease
Society of America. Dr. Tang has been recognized for his extraordinary
expertise in molecular microbiology, diagnosis, and monitoring and has
over 200 peer-reviewed articles and book chapters in this field.

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PRACTICAL GUIDANCE FOR

Practical Guidance for Clinical Microbiology Laboratories:

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Other U.S. and International Guidelines Concerning Speciﬁc Populations and

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EPIDEMIOLOGY AND CLINICAL PRESENTATION OF ACUTE RESPIRATORY VIRAL

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TABLE 1 Classiﬁcation and characterization of common respiratory viruses

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eRhinovirus isolation precautions.

gAirborne transmission may be possible in some cases.

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Close contact in living environments such as long-term care facilities facilitate

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infection (33). In older adults, increased susceptibility to viral infections, age-dependent

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Section Summary and Recommendations

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GUIDELINES ADDRESSING THE DIAGNOSIS AND MANAGEMENT OF SYNDROMES

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Other U.S. and International Guidelines Concerning Speciﬁc Populations and

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be undertaken in a class II BSC included aliquoting specimens, diluting specimens,

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Acute Respiratory Viral Infection following Travel

Section Summary and Recommendations

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investigations (e.g., emerging pathogen investigations and outbreak investigations),

SPECIMEN COLLECTION FOR LABORATORY DETECTION OF ACUTE RESPIRATORY

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Appropriate Specimen Collection Is Critical for Virus Detection in the Laboratory

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TABLE 3 Sensitivity of respiratory viral detection from different specimen typesa

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samples are additionally recommended for enhanced detection.

eSputum sensitivity varies between CoV strains (180).

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Section Summary and Recommendations

LABORATORY DETECTION OF ACUTE RESPIRATORY VIRUSES

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