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Pandemic Influenza
Last updated August 26, 2006
Agent
Laboratory
Testing for Influenza
General
Considerations
Historical
Perspective
Pandemics
of the 20th Century
Lessons
from Past Pandemics
The
Current H5N1 Threat
Vaccine
Development
Use
of Antiviral Agents
Nonpharmaceutical
Interventions
Pandemic
Preparedness Planning
Infection
Control Considerations
References
Note: Information on avian
influenza is
available in the overviews "Avian Influenza (Bird Flu): Agricultural
and Wildlife Considerations" and "Avian Influenza (Bird Flu):
Implications for Human Disease."
Agent
All past influenza pandemics in humans have been
caused by influenza A viruses. General information about influenza A
viruses (not specific to pandemic strains) is presented in the
bullets below.
- Family: Orthomyxoviridae
- Enveloped virions are 80 to 120 nm in diameter,
are 200 to 300 nm long, and may be filamentous.
- They consist of spike-shaped surface proteins, a
partially host-derived lipid-rich envelope, and matrix (M)
proteins surrounding a helical segmented nucleocapsid (6 to 8
segments).
- The family contains five genera, classified by
variations in nucleoprotein (NP and M) antigens: influenza A,
influenza B, influenza C, thogotovirus, and isavirus.
- Genus: Influenzavirus A
- Consists of a single species: influenza A
virus.
- Influenza A viruses are a major cause of
influenza in humans.
- The multipartite genome is encapsidated, with
each segment in a separate nucleocapsid. Eight different
segments of negative-sense single-stranded RNA are present; this
allows for genetic reassortment in single cells infected with
more than one virus and may result in multiple strains that are
different from the initial ones (see References:
Voyles 2002).
- The genome consists of 10 genes encoding for
different proteins (eight structural proteins and two
nonstructural proteins). These include the following: three
transcriptases (PB2, PB1, and PA), two surface glycoproteins
(hemagglutinin [HA] and neuraminidase [NA]), two matrix proteins
(M1 and M2), one nucleocapsid protein (NP), and two
nonstructural proteins (NS1 and NS2).
- The virus envelope glycoproteins (HA and NA) are
distributed evenly over the virion surface, forming
characteristic spike-shaped structures. Antigenic variation in
these proteins is used as part of the influenza A virus subtype
definition (but not used for influenza B or C viruses).
- Influenza A virus subtypes:
- There are 16 different HA antigens (H1 to H16)
and nine different NA antigens (N1 to N9) for influenza A. Until
recently, 15 HA types had been recognized, but a new type (H16)
was isolated from black-headed gulls caught in Sweden and the
Netherlands in 1999 and reported in the literature in 2005 (see
References:
Fouchier 2005).
- Human disease historically has been caused by
three subtypes of HA (H1, H2, and H3) and two subtypes of NA (N1
and N2).
- More recently, human disease has been recognized
to be caused by additional HA subtypes, including H5, H7, and H9
(all from avian origin).
- All known subtypes of influenza A can be found in
birds, and feral aquatic birds are the major reservoir for
influenza A viruses. Feral birds generally do not develop severe
disease from influenza; however, domestic chickens and turkeys
are susceptible to severe and potentially fatal influenza.
- Certain mammals also are susceptible to
influenza. Influenza A viruses have traditionally been known to
cause disease in horses, pigs, whales, and seals; however, the
range of several influenza A subtypes is expanding to further
mammalian species. H5N1 influenza A recently has been shown to
infect cats, leopards, and tigers (see References:
Keawcharoen 2004; Webster 2006). Cases of canine
influenza have been recognized in the United States and are
being caused by H3N8 influenza A, a subtype traditionally
found in horses (see References:
Crawford 2005).
- Influenza A virus subtype strains
- Antigenic strain nomenclature is based on: (1)
host of origin (if other than human), (2) geographic origin, (3)
strain number, (4) year of isolation, and (5) HA and NA type.
(Examples are as follows: A/Hong Kong/03/68[H3N2],
A/swine/Iowa/15/30[H1N1].)
- H5N1 strains have been differentiated into
genetic clades, with nonoverlapping case distributions. All
human H5N1 strains are grouped in clade 1 (see References:
WHO Global Influenza Program Surveillance Network).
- Classification of influenza A strains by pandemic
potential
- Strains from past pandemics:
"Noncontemporary" strains are those from previous pandemics that
pose some degree of risk to the public owing to decreased
immunity in the current population. The term is currently used
to describe strains from the Asian flu (H2N2) but could be
applied to strains from the earlier Spanish flu pandemic (H1N1)
(see References:
CDC: Interim CDC-NIH recommendation for raising the biosafety
level for laboratory work involving noncontemporary human
influenza [H2N2] viruses).
- Nonpandemic strains: These include strains
that have recently circulated or are currently circulating in
the human population (ie, those belonging to H1N1, H3N2, and
H1N2 subtypes).
- Potential pandemic strains: Potential
pandemic strains must have the following features: (1) have an
antigenic makeup to which the population is immunologically
naive, (2) be able to replicate in humans, and (3) efficiently
transmit from human to human. Because of homosubtypic immunity
(see below), new pandemic strains are most likely to be of
subtypes not previously recognized in human populations.
Currently, strains of H5 and H7 subtypes are of greatest
concern.
- Animal pandemic strains (including avian
influenza strains): Animal strains such as H5N1 avian
influenza are not considered human pandemic strains unless the
above criteria are met, but they have significant potential to
evolve into new human pandemic strains through the process of
genetic reassortment (see below) or through gradual adaptation
to the human host. Most avian strains are not of concern as
potential pandemic strains.
- Avian influenza
- The term "avian influenza" is used to describe
influenza A subtypes that primarily affect chickens, turkeys,
guinea fowls, migratory waterfowl, and other avian species.
- "Avian influenza" is an ecological classification
that does not correspond exactly to other classification
schemes.
- As with other influenza A subtypes, standard
nomenclature is used to name strains (eg, A/Chicken/HK/5/98
[H5N1]).
- Avian influenza strains in domestic chickens and
turkeys are classified according to disease severity, with two
recognized forms: highly pathogenic avian influenza (HPAI), also
known as fowl plague, and low-pathogenic avian influenza (LPAI).
Avian influenza viruses that cause HPAI are highly virulent, and
mortality rates in infected flocks often approach 100%. LPAI
viruses are generally of lower virulence, but these viruses can
serve as progenitors to HPAI viruses. The current strain of H5N1
responsible for die-offs of domestic birds in Asia is an HPAI
strain; other strains of H5N1 occurring elsewhere in the world
are less virulent and, therefore, are classified as LPAI
strains. All HPAI strains identified to date have involved H5
and H7 subtypes.
- Human infections have been associated with both
HPAI and LPAI strains (see References:
HHS: Pandemic influenza plan).
- Evidence that HPAI strains arise from LPAI
strains has led the World Organization for Animal Health to
classify all H5 or H7 strains as notifiable (see References:
Alexander 2003, Capua 2004, OIE 2005).
- In the United States, currently only HPAI avian
strains and reconstructed 1918 H1N1 strains are regulated as
select agents (see Biosafety and Biosecurity, below).
- The 1918 influenza pandemic strain (H1N1) appears
to be of avian origin (see References:
CDC: Information about pandemic influenza viruses).
- Physical characteristics of influenza A viruses
- Strains are sensitive to lipid solvents, nonionic
detergents, formaldehyde, and oxidizing agents.
- They are inactivated by ionizing radiation, pH
extremes (>9 or <5), and temperatures greater than 50°C.
- Viruses remain infectious after 24 to 48 hours on
nonporous environmental surfaces and less than 12 hours on
porous surfaces (see References:
Bean 1982). (Note: The importance of fomites in disease
transmission has not been determined.)
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Laboratory
Testing for Influenza
The following statements regarding laboratory testing
apply to influenza viruses in general, not just to influenza testing
in a pandemic setting. During a pandemic, recommendations for
laboratory testing may change, depending on a number of factors,
including availability of testing reagents and laboratory
staffing/surge capacity.
General Considerations
- Tests for influenza virus include viral culture,
polymerase chain reaction (PCR), rapid antigen testing, and
immunofluorescence. Serologic tests are used to retrospectively
diagnose infection.
- Laboratory tests do not need to be conducted on all
patients with suspected influenza. Factors that influence the
decision to test or not test patients with signs and symptoms of
influenza include:
- Residence in a healthcare facility:
Documentation of influenza virus infection in inpatients or
residents of long-term care facilities is important for
detection and control of outbreaks.
- Treatment options: Testing should be
performed if laboratory results influence clinical decision
making.
- Level of influenza activity in the
community: The positive predictive value of influenza tests,
especially rapid assays, increases with prevalence of influenza
in the community; therefore, if the prevalence of influenza is
low, the utility of the tests decreases. As influenza prevalence
increases, the predictive value of clinical diagnosis without
laboratory testing also increases and laboratory confirmation
may not be necessary (see References:
CDC: Interim guidance for influenza diagnostic testing during
the 2004-05 influenza season; Monto 2005).
- Participation in a surveillance program:
Sentinel surveillance can be useful to determine which strains
are circulating in the community and to assess the degree of the
match between circulating viruses and those used to make the
vaccine for that year.
- Patients who meet the criteria for a novel
influenza virus: During a pandemic alert period, patients
who meet certain criteria (such as influenza symptoms and recent
travel to an area affected by a novel strain) should be
considered for laboratory testing.
- Pandemic considerations: As noted above,
recommendations for testing during a pandemic may be somewhat
unique and dependent upon factors such as availability of
reagents and laboratory surge capacity.
- The sensitivity and specificity of laboratory tests
appears to vary with the involved strain, which has implications
for emerging variants (see References:
Weinberg 2005).
- Laboratory tests are required for specific
identification of pandemic strains. The most likely ways that a
pandemic strain would be detected initially are:
- Outbreak investigations or investigation of
unexplained death in a previously healthy individual
- Influenza surveillance with laboratory testing
and characterization of unusual strains
- Investigation of unusual laboratory findings
- State and local health departments should be
prepared to process or test for the following (if they have the
capability, as described below) (see References:
HHS: Pandemic influenza plan).
- Avian influenza A (H5N1) and other avian
influenza viruses
- Other animal influenza viruses
- New or re-emergent human influenza viruses (such
as H2 strains)
- Testing during a pandemic (see References:
HHS: Pandemic influenza plan):
- CDC will update protocols and distribute reagents
as necessary.
- The need for confirmatory testing will diminish
as the pandemic progresses. Some level of continued monitoring
will be necessary to monitor changes in antigenicity and
antiviral susceptibility. CDC will provide appropriate guidance
in such situations.
- Reporting and referral (see References:
HHS: Pandemic influenza plan)
- Clinical laboratories should contact their state
or local health departments if they receive specimens from
patients with possible novel influenza suspected on the basis of
clinical and epidemiologic criteria.
- Public health laboratories should send specimens
to CDC if the patient meets clinical and epidemiologic criteria
and (1) tests positive for influenza A by reverse transcriptase
polymerase chain reaction (RT-PCR) or rapid testing or (2) tests
negative for influenza A by rapid testing and RT-PCR is not
available. Laboratories without capacity for testing avian
strains by indirect immunofluorescence (IFA) or RT-PCR should
send untypable influenza isolates to CDC.
- Any unusual subtype should be reported to CDC
through their emergency response hotline (770-488-7100).
- Laboratory-based influenza surveillance networks
- WHO Global Influenza Surveillance Network (see References)
- CDC National Respiratory and Enteric Virus
Surveillance System (NREVSS) (see References)
- State or local surveillance health department
surveillance networks
Specimen Collection
- Appropriate specimens for testing include: nasal
wash /aspirate, nasopharyngeal swab, throat swab, broncheoalveolar
lavage, tracheal aspirate, pleural fluid tap, sputum, and autopsy
specimens (see References:
HHS: Pandemic influenza plan [Part 2, Supplement 2]).
- Specimens from living patients optimally should be
collected within 4 days after illness onset.
- Some rapid test kits require specific specimen
types and storage/transport methods.
- Nasopharyngeal swabs, nasal washes, and nasal
aspirates are considered to be more sensitive than throat swabs
for culture of most respiratory viruses, including convention
influenza strains, and are preferred for children younger than 2
years of age.
- Pharyngeal swabs collected 4 to 8 days after onset
of illness may be more sensitive for detection of influenza A
(H5N1) than nasal swabs (see References:
WHO: Writing Committee of WHO Consultation on Human Influenza
A/H5l 2005).
- Only sterile Dacron or rayon swabs with plastic
shafts should be used. Calcium alginate swabs or swabs with wooden
sticks should not be used.
- Viral transport media should be used for
nasopharyngeal and oropharyngeal swabs and specimens should be
maintained at refrigerator temperature (4°C to 8oC)
until testing is performed. Freezing at 70°C is best for
maintaining viability during extended storage
- With regard to autopsy specimens, large airways
have the highest yield for immunohistochemistry (IHC) tests. Eight
blocks or fixed-tissue specimens from each of the following sites
should be obtained. Fixed tissue should be transported at room
temperature (not frozen); fresh unfixed tissue should be frozen.
- Central (hilar) lung with segmental bronchi
- Right and left primary bronchi
- Trachea (proximal and distal)
- Representative pulmonary parenchyma from right
and left lung
- Infection control precautions should be observed
during specimen collection.
- Specimen collection procedures for animals have
been described by the World Health Organization (WHO) (see References:
WHO: Manual on animal influenza diagnosis and surveillance).
Biosafety and Biosecurity
- New safety rules and recommendations for influenza
virus will be published in a revised edition of Biosafety in
Microbiological and Biomedical Laboratories (BMBL) (see References:
CDC: Interim CDC-NIH recommendation for raising the biosafety
level for laboratory work involving noncontemporary human
influenza [H2N2] viruses; CDC: Update on influenza A [H5N1] and
SARS: Interim recommendations for enhanced U.S. surveillance,
testing, and infection controls; HHS: Pandemic influenza plan).
Current recommendations for interpandemic and pandemic alert
periods include:
- Culture of influenza subtypes H1-4, H6, and H8-15
(with exceptions noted below) and culture of specimens from
patients not suspected of having novel influenza strains
requires BSL-2 containment and practices (Animal BSL-2 for
animal models).
- Culture of noncontemporary influenza strains
(H2N2) or research involving reverse genetics of the 1918
Spanish flu strain (H1N1) requires BSL-3 facilities and Animal
BSL-3 practices, including containment with rigorous adherence
to additional respiratory protection and clothing change
protocol, use of negative pressure, high-efficiency particulate
air (HEPA) filtered respirators or positive air-purifying
respirators (PAPRs), use of HEPA filtration for treatment of
exhaust air, and amendment of personnel practices to include
personal showers prior to exiting the laboratory.
- Culture from patients suspected of having avian
influenza, other novel influenza strains, or severe acute
respiratory syndrome (SARS) coronavirus should only be conducted
under enhanced BSL-3 containment (also see Biosecurity below).
This includes controlled access, double-door entry with changing
room and shower, use of respirators, decontamination of all
waste, and showering out of all personnel. These diagnostic
activities must be kept separate from routine influenza
diagnostic activities (eg, probably H1 or H3) to prevent
recombination.
- IFA of specimens requires BSL-2 containment and
practices. Culture biocontainment recommendations should be
implemented when IFA is used for culture identification.
- Direct detection methods, including commercial
antigen detection assays and RT-PCR, should be conducted under
BSL-2 with a Class II biological safety cabinet. Serologic
methods require BSL-2 containment.
- If H5N1 avian influenza virus is presumptively
identified by one of the above direct methods, further work
should be conducted using the enhanced BSL-3 procedures
described for culture.
- Any new or re-emergent human influenza strain
with suspected pandemic potential should be treated in the same
manner as described for H5N1 avian influenza.
- Additional requirements and recommendations apply
for laboratory work involving live animals.
- Biosecurity
- Human influenza strains, with a few exceptions
(see below), are not regulated as select agents. Inclusion of
potentially pandemic strains on the select agent list is
currently under consideration (see References:
CDC: Interim CDC-NIH recommendation for raising the biosafety
level for laboratory work involving noncontemporary human
influenza [H2N2] viruses; CDC: Update on avian influenza A[H5N1]
and SARS). Despite the absence of regulatory authority, standard
biosecurity measures should be maintained for potentially
pandemic strains.
- The US Department of Agriculture (USDA)
classifies highly pathogenic avian influenza (HPAI) as an
agricultural select agent regulated under 7 CFR part 331 and 9
CFR Part 121 of the Federal Register, which was published
as a Final Rule in the March 18, 2005, issue (see References:
USDA/APHIS: Agricultural Bioterrorism Protection Act of 2002).
Laboratories that work with HPAI strains (H5 or H7) or perform
diagnostic cultures for suspected human cases of avian influenza
caused by H5 or H7 or suspected cases of SARS must be registered
with the USDA.
- Both registered and exempt laboratories that
identify a select agent contained in a specimen presented for
diagnosis, verification, or proficiency testing must secure the
agent against theft, loss, or release until transfer or
destruction. Unregistered laboratories must transfer or destroy
select agents within 7 days of identification. Any theft, loss,
or release of the agent must be reported to the select agent
authority (see References:
USDA/APHIS: Questions and answers).
- Effective October 20, 2005, "reconstructed
replication competent forms of the 1918 pandemic influenza virus
containing any portion of the coding regions of all eight gene
segments" will be regulated as select agents under an interim
rule from the US Department of Health and Human Services (HHS)
(see References:
CDC: Select Agent Program).
Virus Isolation by Cell Culture
- Virus isolation is considered the "gold standard"
of influenza testing (see References:
Hayden 2002, Treanor 2005).
- Cell culture measures growth rather than the
presence or absence of specific targets. As cell lines are
designed to support the growth of a wide range of viruses, cell
culture will likely allow for detection of emerging and pandemic
influenza strains (see References:
Australian Government Department of Health and Ageing).
- Isolates obtained from cell culture are required
for strain characterization, which is an integral part of global
influenza surveillance and monitoring activities during a pandemic
(see References:
HHS: Pandemic influenza plan).
- Cell culture is subject to certain restrictions
(see Biosafety
and Biosecurity above).
- Specimens for culture optimally should be collected
within 3 days after illness onset.
- Turnaround time for the standard method is 2 to 14
days.
- Culture consists of growth on a cell monolayer,
detection of viral growth, and specific identification.
- Virus detection and identification methods for
standard culture include the following:
- Cell lines include Madin-Darby canine kidney
(MDCK), primary rhesus monkey kidney (PRMK), or cynomolgus
monkey kidney. Other cell lines, such as Vero, mink lung, and
MRC-5, also support growth of influenza virus if trypsin is
incorporated into serum-free medium.
- Cytopathic effect (CPE) is not a
consistent feature of influenza A virus. If present, CPE is
nonspecific, including vacuolization or cell degeneration.
- Assays for haemadsorption (HAd) (ie,
influenza-infected cells bind red blood cells [RBCs]) are
performed blindly, typically at 7 and 14 days or on cells
exhibiting CPE. Other viruses, such as parainfluenza virus and
mumps virus, may also cause HAd. The lack of HAd specificity may
be an advantage in detecting new or pandemic strains.
- Hemagglutination inhibition (HI or HAI) is used
to identify the viral subtype. Cell supernatant is mixed with
RBCs; identification is by quantitative inhibition of
agglutination using subtype-specific antisera. Homologous
strains yield high HI titers. New pandemic strains would likely
be HAd-positive with or without CPE, with low or negative titers
to group-specific antisera.
- Identification of infected cells is by direct or
indirect immunofluorescence (eg, DFA, IFA), enzyme-linked
immunoassays (EIA), or PCR-based methods. Assays with more
conserved, less specific targets are more likely to detect newly
emerged strains.
- The time to detection in culture, as measured in
one study conducted during two influenza seasons, ranged from 5
days (>90% of positive specimens) to 7 days (100% of positive
specimens) (see References:
Newton 2002).
- A golden rule of laboratory testing is to never
process clinical specimens from humans and swine (and presumably
birds) in the same laboratory (see References:
WHO recommended laboratory tests to identify influenza A/H5 in
specimens from patients with influenza-like illness).
- Shell vial assay (rapid culture), when combined
with a rapid detection/identification method, offers a sensitive
and rapid diagnostic alternative to standard culture. This method
does not result in an adequate viral titer or volume for further
characterization and would thus not be appropriate for pandemic
influenza surveillance without subculture.
Direct Detection Methods
Direct detection methods do not result in production
of an isolate and would be inadequate for surveillance or definitive
characterization of pandemic strains. Nevertheless, owing to their
relatively rapid turnaround time, safety, and stability, direct
detection methods play an important role in pandemic influenza
preparedness.
- Reverse transcription PCR (RT-PCR) assays
- The sensitivity of RT-PCR has been reported to be
in the range of 90% to 100% when compared with cell culture.
However, several researchers have reported significantly higher
numbers of total positive specimens with RT-PCR, possibly
reflecting its ability to detect nonviable virions (see References:
Coiras 2003, Hayden 2002, Herrmann 2001, Pachucki 2004, Wallace
1999).
- On February 3, 2006, the Food and Drug
Administration (FDA) announced clearance of an Influenza A/H5
(Asian Lineage) Virus Real-Time Reverse Transcription–Polymerase
Chain Reaction (RT-PCR) Primer and Probe Set and inactivated
virus as a source of positive RNA control for the in vitro
detection of highly pathogenic influenza A/H5 virus (Asian
lineage) (see References:
CDC 2006: New laboratory assay for diagnostic testing of avian
influenza A/H5 [Asian lineage]). These reagents and assay
protocols have been distributed by CDC to state and city LRN
(Laboratory Response Network) laboratories. Testing with the new
assay is limited to LRN-designated laboratories.
- While culture of specimens from possible avian
influenza (H5N1) cases is not recommended without strict
containment and specific registration (described above), RT-PCR
can be conducted using BSL-2 facilities and practices (see References:
HHS: Pandemic influenza plan).
- Common PCR targets include the matrix (M) protein
(for genus-level identification), hemagglutinin, and
neuraminidase (for subtype-level identification). PCR generally
is not used for strain-level identification, which is based on
serologic markers.
- The likelihood that a RT-PCR assay will detect
new pandemic strains increases when more conserved target
sequences are used.
- As with other PCR-based assays, efforts should be
made to minimize and detect amplicon contamination.
- Samples positive by RT-PCR for a novel influenza
subtype should be forwarded to a public health laboratory (if
testing was conducted at a private laboratory) or to CDC for
confirmation (see References: HHS Pandemic influenza plan).
- A molecular microarray for influenza typing and
subtyping using a flow-thru chip platform has been described
(see References:
Kessler 2004).
- The development of portable real-time platforms
has made possible the use of PCR assays in the field (see References:
Perdue 2003).
- Immunofluorescence
- IFA methods may be used to identify influenza to
the species level (influenza A or B) or specific H subtypes
(including H5) directly from specimens or cell culture. CDC
distributes IFA typing and subtyping reagents to
WHO-collaborating laboratories, including many health department
laboratories. If HPAI strains are suspected, enhanced BSL-3
containment should be used (see References:
WHO: Recommended laboratory tests to identify avian influenza A
virus in specimens from humans; FDA: Cautions in using rapid
tests for detecting influenza A viruses; HHS: Pandemic influenza
plan)
- Direct immunofluorescence (DFA) methods are
faster and less labor intensive than IFA but are less sensitive
and are currently only available for genus-specific detection
(see other rapid direct tests in the next bullet).
- Other rapid direct tests (see References:
Call 2005; CDC: Interim guidance for influenza diagnostic testing
during the 2004-05 influenza season; FDA: Cautions in using rapid
tests for detecting influenza A viruses; HHS: Pandemic influenza
plan; Treanor 2005; WHO: WHO checklist for influenza pandemic
preparedness planning).
- Rapid tests detect viral antigen (generally
nucleoprotein) or enzymatic activity (neuraminidase) directly on
patient specimens using a variety of platforms.
- Rapid tests are designed to identify influenza A
only, influenza A or B without identifying the type, or
influenza A or B with type-specific identification.
- Reported sensitivities range from 40% to 80%.
- Sensitivity is generally greater in children than
adults.
- Sensitivity is greater early in the course of
illness.
- Rapid test predictive value and disease
prevalence: The predictive value of rapid assays without
confirmation by a reference test is strongly correlated with
disease prevalence in the community, as is clinical diagnosis
without laboratory testing. When the disease prevalence is low,
the tests' positive predictive value decreases and positive
results should be confirmed by culture or RT-PCR. When influenza
is known to be circulating (ie, high prevalence in the
community), the negative predictive value is lower and
clinicians should consider confirming negative tests with viral
culture or other tests.
- Rapid test predictive value and diagnostic
indications: Rapid tests increase the diagnostic predictive
value when used for confirmation of influenza (when symptoms are
strongly suggestive) and for ruling out influenza (when symptoms
suggest illness other than influenza). When symptoms are not
strongly suggestive in either direction, the utility of rapid
testing becomes questionable.
- While the sensitivity and specificity of rapid
tests has been evaluated for circulating strains, these measures
are largely unknown for detection of emerging strains (including
pandemic strains) (see References:
FDA: Cautions in using rapid tests for detecting influenza A
viruses). Only 4 (36%) of 11 culture-positive H5N1 influenza A
specimens from patients in Thailand were positive by rapid
antigen tests (see References:
WHO Writing Committee of WHO Consultation on Human Influenza
A/H5 2005).
- WHO, in their Checklist for Influenza Pandemic
Preparedness Planning, recommends against routine use of
commercial rapid antigen detection kits and suggests they be
used for outbreak investigation only when no other options exist
(see References:
WHO Writing Committee of WHO Consultation on Human Influenza
A/H5 2005).
- During a pandemic, rapid tests may be useful for
distinguishing influenza from other respiratory illnesses (see
References:
HHS: Pandemic influenza plan).
Serology
- Serologic testing can be used for retrospective
diagnosis of infection but is rarely useful for patient management
and is not widely available. However, serology may be useful for
investigation of novel viruses (see References:
Hayden 2002; Treanor 2005; HHS: Pandemic influenza plan).
- Acute-phase sera should be collected within 1 week
after illness onset, and convalescent sera should be collected 2
to 3 weeks later.
- The most common serologic methods are complement
fixation (CF), HAI, and enzyme immunoassays (EIA). A variety of
other methods, such as neutralization, microneutralization, single
radial hemolysis, radial immunodiffusion, and Western blot, have
been reported (see References:
Hayden 2002, Rowe 1999).
- IgG, IgA, and IgM antibodies appear simultaneously
about 2 weeks after initial infection. Antibodies appear more
quickly with subsequent infections. Tests for IgM and IgA are less
useful than IgG for routine clinical use, as most infections are
reinfections (see References:
Australian Government Department of Health and Ageing; Hayden
2002)
- Peak antibody response occurs 4 to 7 weeks after
infection.
- Since most people are repeatedly exposed to
influenza viruses, a fourfold rise in titer between acute and
convalescent sera generally is considered necessary for
confirmation of influenza infection.
- While paired sera are optimal, single convalescent
specimens may be useful in investigations involving novel viruses.
Antibody test results have been compared with results from
age-matched persons in the acute phase of illness or from non-ill
controls. The geometric mean titers between the two groups to a
single influenza virus type or subtype can be compared (see References:
HHS: Pandemic influenza plan)
- HAI EIAs measure antibody to hemagglutinin. These
tests are more sensitive than CF, but their increased specificity
appears to limit their ability to detect new strains.
- HAI titers of at least 1:40 or serum neutralizing
titers of 1:8 or greater are associated with protection.
- HAI titers in human avian influenza cases have been
low or undetectable (see References:
HHS: Pandemic influenza plan).
- CF measures antibody response to nucleoprotein,
which is conserved among influenza A strains. This feature could
be an advantage for diagnosis of infection with novel pandemic
strains.
- The microneutralization assay can sensitively and
specifically detect H5N1 antibody in patients with H5N1 influenza.
Since the test uses infectious organisms, HPAI strains should be
tested under enhanced BSL-3 containment. As with other tests,
paired sera are preferable to single specimens (see References:
HHS: Pandemic influenza plan).
Susceptibility Testing
- Susceptibility testing generally is conducted at
specialized laboratories as part of surveillance or research and
is considered an integral component of pandemic influenza
response.
- Plaque reduction assay (see References:
Hayden 1980, McKimm-Breschkin 2003)
- The traditional influenza susceptibility testing
method for the M2 ion channel inhibitors (amantadine,
rimantadine)
- Can detect a wide range of resistance phenotypes
- Limited utility for neuraminidase inhibitors
- Enzyme inhibition assays (see References:
McKimm-Breschkin 2003, Wetherall 2003)
- Useful for assay of neuraminidase inhibitors
- Chemiluminescent or fluorescent substrates
- Sequence analysis (see References:
McKimm-Breschkin 2003, Wetherall 2003)
- Used to detect mutations in genes known to be or
suspected of being responsible for resistance
- Neuraminidase gene sequences from strains
isolated prior to introduction of the drugs can be used to
evaluate current strain sequences
- Mutations in the M2 can be used to detect
amantadine resistance (see References:
Pachucki 2004)
- The Neuraminidase Inhibitor Susceptibility Network
(NISN) was established to monitor susceptibility of clinical
isolates to zanamivir and oseltamivir. The chemiluminescent
neuraminidase enzyme assay was chosen by the NISN as the method of
choice for testing neuraminidase inhibitors (see References:
Wetherall 2003).
Laboratory Values That May Trigger Concern for Human Pandemic
Influenza
- Positive test for influenza from a patient with
risk factors for avian influenza
- Culture: CPE positive or negative; HAd positive; HI
titer low or negative and no other hemagglutinating viruses
identified
- RT-PCR positive for H5 or H7
- RT-PCR positive for influenza A from a conserved
target, such as matrix protein, and negative for H1-H3
- A four-fold rise in H5-specific antibody titer
(acute and convalescent serum samples)
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General Considerations
Cross-Immunity
In general, the degree of immunity induced by one
strain of influenza virus to a second challenge with another
influenza virus is related to the taxonomic distance between the two
strains (see References:
Epstein 2003). Several terms that characterize the type of immunity
are identified below.
- Heterologous immunity: Immunization
with one type of influenza virus (eg, A, B, or C) does not offer
protection from challenge with a different type.
- Heterosubtypic immunity (also referred to as
"heterotypic immunity"): Immunization with one
influenza A virus subtype (eg, H1N1) may offer some protection
from challenge with a second influenza A subtype (eg, H5N2). The
degree of protection, or lack of protection, is important to the
discussion of pandemic influenza and vaccine development.
- Homosubtypic immunity: Immunization
with one strain within a subtype (eg, A/Hong Kong/03/68[H3N2])
will frequently offer some protection against challenge with a
second strain within the same subtype (eg,
A/Fujian/447/2003[H3N2]).
- Homologous immunity: Immunization with one
strain of influenza A virus (eg, A/Fujian/447/2003[H3N2]) offers
protection from a second challenge with the same strain.
Antigenic Drift vs Antigenic Shift
- "Antigenic drift" refers to the process of small
genetic changes that influenza viruses continuously undergo from
year to year, which necessitates the development of new vaccines
annually. Partial immunologic cross-reactivity between new strains
and those they are replacing (ie, homosubtypic immunity) limits
morbidity, mortality, and spread in the population.
- "Antigenic shift" refers to substantial genetic
changes caused by the process of genetic reassortment. Relatively
few lineages of influenza A are circulating among humans at any
one time, which reduces the likelihood of significant genetic
reassortments. However, antigenic shift can occur between human
and animal strains, which is what happened with the pandemic
strains of 1957 and 1968. It is important to note that not all
pandemic strains arise from genetic reassortment. For example, the
1918 pandemic strain apparently did not originate through a
reassortment event; rather, it is likely that an avian strain
initially infected humans and then adapted gradually to the human
population over time to become a pandemic strain (see References:
Taubenberger 2005).
Features of Pandemic Strains
Pandemics occur when a novel influenza strain emerges
that has the following features:
- Highly pathogenic for humans
- Easily transmitted between humans
- Genetically unique (ie, lack of preexisting
immunity in the human population)
Pandemic Phases
In reviewing the public health implications of a
pandemic, it is useful to understand the various phases that a
pandemic will likely go through. These are outlined in the following
table. (Note: In 1999, WHO developed a set of pandemic phases; these
were revised in the new WHO Global Influenza Preparedness Plan that
was released in April 2005. The phases identified below are from the
2005 Plan [see References:
WHO: WHO global influenza preparedness plan 2005].) The current
pandemic phase for H5N1 is Phase 3.
|
Phase |
Characteristics of Phase |
Rationale |
|
Phase 1 |
No new influenza virus subtypes have been
detected in humans. An influenza virus subtype that has caused
human infection may be present in animals. If present in
animals, the risk of human infection or disease is considered
to be low. |
It is likely that influenza subtypes that
have caused human infection and/or disease will always be
present in wild birds or other animal species. Lack of
recognized animal or human infections does not mean that no
action is needed. Preparedness requires planning and action in
advance. |
|
Phase 2 |
No new influenza virus subtypes have been
detected in humans. However, a circulating animal influenza
virus subtype poses a substantial risk of human disease.
|
The presence of animal infection caused by
a virus of known human pathogenicity may pose a substantial
risk to human health and justify public health measures to
protect persons at risk. |
|
Pandemic Alert Period
|
|
Phase 3 |
Human infection(s) with a new subtype, but
no human-to-human spread, or at most rare instances of spread
to a close contact. |
The occurrence of cases of human disease
increases the chance that the virus may adapt or reassort to
become transmissible from human to human, especially if
coinciding with a seasonal outbreak of influenza. Measures are
needed to detect and prevent spread of disease. Rare instances
of transmission to a close contact, for example, in a
household or healthcare setting, may occur but do not alter
the main attribute of this phase (ie, that the virus is
essentially not transmissible from human to human). |
|
Phase 4 |
Small cluster(s) with limited
human-to-human transmission but spread is highly localized,
suggesting that the virus is not well adapted to humans.
|
Virus has increased human-to-human
transmissibility but is not well adapted to humans and remains
highly localized, so that its spread may possibly be delayed
or contained. |
|
Phase 5 |
Larger cluster(s) but human-to-human spread
is still localized, suggesting that the virus is becoming
increasingly better adapted to humans but may not yet be fully
transmissible (substantial pandemic risk). |
Virus is more adapted to humans and
therefore more easily transmissible among humans. It has
spread in larger clusters, but spread is localized. This is
likely to be the last chance for massive coordinated global
intervention, targeted to one or more foci, to delay or
contain spread. In view of possible delays in documenting
spread of infection during pandemic Phase 4, it is anticipated
that there would be a low threshold for progressing to Phase
5. |
|
Pandemic Period |
|
Phase 6 |
Increased and sustained transmission among
general population. |
Major change in global surveillance and
response strategy, since pandemic risk is imminent for all
countries. The national response is determined primarily by
the disease impact within the country. |
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Historical Perspective
Earliest reports of influenza epidemics date back to
412 BC and were recorded by Hippocrates. A number of epidemics that
likely were influenza were described in the Middle Ages, and one
that was probably a true pandemic took place in 1510 (see References:
Beveridge 1978). Other key historical facts include the following:
- One of the earliest recorded pandemics occurred in
1580. Like the 1918 pandemic, this one was particularly severe. It
started in Asia and spread to Africa, Europe, and the Americas. In
6 weeks it afflicted all of Europe. Death rates were high; 9,000
of 80,000 people died in Rome, and some Spanish cities were
described as "nearly entirely depopulated" by the disease (see References:
Beveridge 1978).
- Ten pandemics have been recorded in the past 300
years. During this time, 10 to 49 years has occurred between
pandemics with an average of 24 years.
- During the 17th century, localized epidemics were
reported, and in the 18th century at least three pandemics
occurred (1729-30, 1732-33, and 1781-82).
- Three influenza pandemics occurred during the 19th
century (1830-31, 1833-34, and 1889-90). The 1889 pandemic known
as the Russian Flu began in Russia and spread rapidly throughout
Europe. It reached North America in December 1889 and spread to
Latin America and Asia in February 1890. About 1 million people
died in this pandemic.
Global influenza surveillance was established in 1947
by WHO to better understand the epidemiology of influenza and to
obtain isolates in a systematic fashion for annual vaccine
development (see References:
Hampson 1997).
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Pandemics of the 20th
Century
Three pandemics occurred during the 20th century,
caused by an H1, an H2, and an H3 strain. These are outlined in the
table below and then briefly summarized. Currently, H1 and H3
influenza strains are circulating in the human population.
Scientists have raised concern about the possibility of H2N2
reemerging (also referred to as recycling) in humans, particularly
through accidental release of a laboratory strain (see References:
Dowdle 2006).
|
Date |
Strain |
Estimated No. of Deaths in US |
Comments |
|
1918-19 (Spanish Flu) |
H1N1 |
500,000 |
Global mortality may have been as high as
100 million. The virus likely originated in the US and then
spread to Europe. |
|
1957-58
(Asian Flu) |
H2N2 |
60,000 |
The virus was first identified in China;
approximately 1 million people died globally during this
pandemic. |
|
1968-69
(Hong Kong Flu) |
H3N2 |
40,000 |
The death rate from this pandemic may have
been lower because the strain had a shift in the hemagglutinin
(H) antigen only and not in the neuraminidase (N) antigen.
|
1918-19 (Spanish Flu)
This pandemic was caused by an influenza A (H1N1)
strain. Worldwide, about one third of the world's population was
infected and had clinically apparent illness (about 500 million
people) and an estimated 50 to 100 million died (see References:
Johnson 2002, Taubenberger 2006). E | |
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