Pandemic Influenza

Last updated April 29, 2008

Agent
Laboratory Testing for Influenza
General Considerations
Historical Perspective
Pandemics of the 20th Century
Lessons from Past Pandemics
The Pandemic Severity Index
The Current H5N1 Threat
Vaccine Development
Use of Antiviral Agents
Community Mitigation Strategies
Pandemic Preparedness Planning
Hospital Pandemic Preparedness Planning
Infection Control Considerations
References

Note: Information on avian influenza is available in the overviews "Avian Influenza (Bird Flu): Implications for Human Disease" and "Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations" in the Avian Flu section of this site.

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 and subclades 1 through 3 of clade 2 (see References: WHO 2007: Antigenic and genetic characteristics of H5N1 viruses and candidate H5N1 vaccine viruses developed for potential use as pre-pandemic vaccines).
• 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.
• During a pandemic, recommendations for laboratory testing may be somewhat unique and dependent upon factors such as: (1) availability of reagents and laboratory surge capacity, (2) the presence or absence of other influenza strains in the community, (3) level of influenza activity in the community, and (4) treatment considerations.
• 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
• Testing of persons with influenza-like symptoms who meet certain exposure criteria
• 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

The following information is taken from a field operations guide for H5N1 influenza that was released by WHO in early November 2006 (see References: WHO 2006: Collecting, preserving, and shipping specimens for the diagnosis of avian influenza A [H5N1] virus infection). Even though the guide is specific to H5N1 influenza, the information provided would likely be applicable to other pandemic strains. Information also was taken from the HHS Pandemic Influenza Plan where noted (see References: HHS 2005: Pandemic influenza plan [Part 2, Supplement 2]).

Specimens to collect from suspect cases

• Upper respiratory tract
• Posterior-pharyngeal (throat) swabs (provide the highest yield)
• Nasal swabs with nasal secretions (from the anterior turbinate areas) or nasopharyngeal aspirates or swabs (these specimens are more appropriate for seasonal influenza and the yield may be lower for avian influenza)
• Lower respiratory tract:
• A tracheal aspirate or bronchoalveolar lavage specimen (if the patient is intubated)
• Blood:
• Serum (acute and convalescent if possible)
• Secondary specimens (to be collected as indicated early in a pandemic or under specific circumstances):
• Plasma in EDTA (for detection of viral RNA)
• Rectal swab (for patients with diarrhea, if the strain produces diarrhea)
• Spinal fluid (if the strain produces meningitis and a spinal tap is performed for diagnostic purposes)
• Pleural tap fluid (referred to in the HHS Plan)
• Autopsy specimens (referred to in the HHS Plan)

When to collect specimens from suspect cases

• Ideally, a throat swab should be taken within 3 days after illness onset; if initial specimens are negative, but a high index of suspicion remains, testing should be repeated as soon as possible. (According to the HHS Plan, specimens optimally should be collected within 4 days after illness onset.)
• Virus may be detected in tracheal aspirates from onset of lower respiratory symptoms until the second or third week of illness.
• An acute phase serum sample should be taken 7 days or less after symptom onset and a convalescent sample should be taken 3 to 4 weeks following illness onset.
• Single serum samples should be collected 14 days or later after symptom onset.
• Serum or plasma for detecting viral RNA should be obtained during the first 7 to 9 days after symptom onset.
• Ideally specimens should be collected before antiviral therapy, but treatment should not be delayed in order to take specimens.
• Specimens should be collected from deceased patients as soon as possible after death.

Specimen collection and transport

• Detailed methods for specimen collection and transport are provided in the WHO field guide (see References: WHO 2006: Collecting, preserving, and shipping specimens for the diagnosis of avian influenza A (H5N1) virus infection).
• Infection control precautions should be consistently observed during specimen collection.
• Only sterile dacron or rayon swabs with plastic shafts should be used. Calcium alginate or cotton swabs or swabs with wooden sticks should not be used (or used only when appropriate swabs are not available).
• Viral transport media (VTM) should be used for nasopharyngeal and oropharyngeal swabs and, according to the HHS Plan, specimens should be maintained at refrigerator temperature (4ºC to 8^oC) until testing is performed. Freezing at 70ºC is best for maintaining viability during extended storage.
• According to the HHS Plan, 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

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).
• 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).
• Molecular microarray tests using flow-through chip technology
• A molecular microarray for influenza typing and subtyping using a flow-thru chip platform was initially described in 2004 (see References: Kessler 2004).
• Two reports released in August 2006 involved a study of the FluChip-55 diagnostic microarray and showed that the test could be a valuable tool in identifying influenza viruses (see References: Mehlmann 2006, Townsend 2006). The FluChip used in the study contained 55 sequences of RNA representing a variety of type A and type B flu viruses, including H3N2, H1N1, and H5N1. Combined results after two rounds of testing showed that the FluChip allowed users to obtain correct information about both type and subtype from 72% of 72 samples tested. Full information on type, but only partial information on subtype, was obtained for an additional 13% of the samples, while 10% of the samples could be identified by type only (no information about subtype). The entire analysis time was less than 12 hours.
• Scientists recently have developed an improved microarray test referred to as the “MChip,” which has several advantages over the FluChip. While the FluChip is based on three influenza genes—hemagglutinin (HA), neuraminidase (NA) and matrix (M)—the MChip is based on only the M gene segment, which mutates much less rapidly. A recent evaluation demonstrated that the assay exhibited a clinical sensitivity of 97% and clinical specificity of 100% (see Nov 15, 2006, CIDRAP News Story).
• 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)
• Researchers have recently reported a PCR assay to efficiently and accurately detect oseltamivir-sensitive and oseltamivir-resistant H5N1 strains (see References: Suwannakarn 2006). The assay is based on the fact that oseltamivir resistance is caused by a single amino acid substitution from histidine (H) to tyrosine (Y) at position 274 of the neuraminidase active site.
• 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.

WHO Pandemic Phases
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.
From WHO: WHO global influenza preparedness plan 2005 (see References).

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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, Patterson 1986).
• Ten pandemics have been recorded in the past 300 years (see References: Osterholm 2007: The fog of pandemic preparedness). The time between starting points of these pandemics has ranged from 10 to 49 years, with an average of 24 years.
• During the 17th century, localized epidemics were reported, and in the 18th century at least two pandemics occurred (1732-33, and 1781-82).
• Five influenza pandemics occurred during the 19th century (1800-02, 1830-33, 1847-48, 1857-58, and 1889-90) (see References: Osterholm 2007: The fog of pandemic preparedness). 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).

Influenza Pandemics of the 20th Century: Impact in the United States*
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.
*All three pandemics were characterized by a shift in age distribution of deaths to younger populations under age 65 (at least initially); shift was particularly dramatic during the 1918 pandemic (see References: HHS:Q&A; HHS: Influenza pandemics; Kilbourne 2006; Simonsen 2004; Webster 1997).

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). Earlier estimates implied that the death toll was 20 to 40 million, but more recent evidence supports the higher figures. Adjusting for today's population, a similar pandemic would yield a modern death toll of 175 to 350 million.

• One recent study projected 51 to 81 million deaths using 2004 population estimates; however, the authors assumed wide variability in death rates by country based on per-capita income and other factors (see Dec 22, 2006 CIDRAP News Story and see References: Murray 2006).
• Another recent report suggests that if a 1918-like pandemic were to occur with increased deaths in the elderly population, over 142.2 million people would die and there would be a GDP loss of US $4.4 trillion worldwide (see References: Osterholm 2007: Unprepared for a pandemic).
• Some have suggested that the death toll from a similar pandemic occurring in modern times would be lower owing to improved medical care and public health infrastructure (see References: Morens 2007); however, if attack rates were high, medical and public health systems could quickly become overwhelmed.

The 1918 pandemic began with a relatively mild "herald" wave in the spring of 1918. During that time, outbreaks were reported in Europe and in the United States (particularly in military training camps for new recruits headed to the war in Europe) (see References: Reid 2001, Glezen 1996).

• Many investigators believe that the strain originated in the United States (perhaps in rural Kansas) and then migrated initially to France before spreading throughout Europe (see References: Barry 2004). However, others believe that the strain may have been circulating in the Mid-Atlantic States as early as February of 1918 (see References: Simonsen 2004). Furthermore, an outbreak of severe respiratory disease occurred in an army camp in France in 1916-17 (see References: Oxford 2000). A significant clinical feature of the disease was cyanosis, which also was a predominant finding among those who acquired the pandemic strain of influenza. It is possible that this outbreak represented H1N1 infection and was an early precursor to the pandemic. At any rate, it is clear that the 1918-19 pandemic did not begin in Asia, although the origin of the implicated H1N1 strain still remains a mystery.
• This first wave was followed by two additional waves in the fall and winter of 1918-19 that were much more severe (see References: Taubenberger 2006). The second highly virulent wave spread rapidly around the world in the fall of 1918; it took only 2 months for the pandemic to circle the globe at that time.
• Recorded case-fatality rates varied around the globe. In the US military, death rates ranged from 5% to 10% (see References: Barry 2004). Higher rates were reported in some areas.
• Additional waves that were not as severe occurred in 1919 and 1920.

An unusual feature of the pandemic was the age-related mortality; the pandemic strain killed a disproportionate number of healthy young adults. This led to the observation of a "W" shaped age-related mortality curve in the United States, with high rates of mortality among very young children, persons 15 to 45 years of age, and the elderly (see References: Reid 2001; Glezen 1996; Morens 2007). Usually the curve associated with influenza mortality follows a "U" shape, with excess deaths occurring only among the very young and the elderly. One striking feature of the pandemic was its impact on pregnant women; a summary of 13 studies involving pregnant women demonstrated that case-fatality rates ranged from 23% to 71% (see References: Barry 2004).

The excess influenza deaths appear to have involved two overlapping clinical-pathologic syndromes (see References: Morens 2007). One pattern was aggressive bronchopneumonia, most likely caused by a secondary bacterial pneumonia. The second pattern was a rapidly evolving severe acute respiratory distress-like syndrome (ARDS).

In October 2005, CDC reported that scientists had reconstructed the 1918 pandemic H1N1 strain and tested it in mice (see References: Tumpey 2005). They found that mice infected with the 1918 strain died in as little as 3 days, and mice that survived as long as 4 days had 39,000 times as many virus particles in their lungs as did mice infected with a control influenza virus, a Texas strain of H1N1 from 1991. All the mice infected with the 1918 virus died, while those exposed to the Texas strain survived. Further, the 1918 virus was at least 100 times as lethal as an engineered virus that contained five 1918 genes and three genes from the Texas H1N1 strain. The researchers found that the mice had severe inflammation in their lungs and bronchial passages, findings very similar to those in people who died of the 1918 virus.

Earlier studies in mice using genetically engineered influenza strains similar to the H1N1 1918 pandemic strain suggest that macrophage activation with high levels of cytokine production may have been a key factor in lung damage caused by the pandemic strain (see References: Kobasa 2004). Investigators have postulated that an overly robust immune response inducing a "cytokine storm" may have contributed to the high case-fatality rates seen in younger populations during the 1918 pandemic. Another study recently found that cynomolgus macaques had an atypical host response to infection with the 1918 virus (characterized by dysregulation of the antiviral response), suggesting that the 1918 virus was able to modulate the host immune response (see References: Kobasa 2007).

Recent genetic sequencing of the 1918 strain indicates that the strain was of avian origin and that the strain did not reassort with a human strain (unlike later pandemics), but rather gradually adapted to humans until it could be efficiently transmitted person to person (see References: Taubenberger 2005). Current evidence indicates that the 1918 virus was an avian-like virus derived in toto from an unknown source (see References: Taubenberger 2006). A two-amino acid change in the hemagglutinin of the 1918 virus was recently shown to abolish transmission among ferrets, confirming the essential role of hemagglutinin receptor specificity for the transmission of influenza viruses in mammals (see References: Tumpey 2007).

1957-58 (Asian Flu)

The Asian flu was caused by an H2N2 strain and originated in China. The virus was initially isolated in Singapore in February 1957 and in Hong Kong in April of that year. The pandemic spread to the Southern Hemisphere during the summer of 1957 and reached the United States in June 1957 (see References: Glezen 1996). The pandemic strain acquired three genes from the avian influenza gene pool in wild ducks by genetic reassortment and obtained five other genes from the then-circulating human strain.

About 69,800 people in the United States died and mortality was spread over three seasons. Overall, the highest mortality rates occurred among the elderly; however, during the initial season in 1957, nearly 40% of the influenza deaths occurred among persons less than 65 years of age (see References: Simonsen 2004). The high case-fatality rate in this age-group declined in subsequent years. Globally, approximately 1 million people died during this pandemic.

1968-69 (Hong Kong Flu)

The Hong Kong flu was caused by an H3N2 strain. The strain acquired two genes from the duck reservoir by reassortment and kept six genes from the virus circulating at the time in humans.

During the pandemic, about 33,800 people died in the United States. 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. Although antibodies to neuraminidase antigen do not prevent infection, they may modify the severity of disease (see References: Glezen 1996). Also, an H3 strain had apparently circulated in the United States around the turn of the century, so elderly persons may have had some protective antibody from past exposure to an H3 strain (see References: Simonsen 2004). This could have caused a lower fatality rate in the elderly.

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Lessons from Past Pandemics

In a recent report issued in January 2005, WHO officials identified key lessons from the three pandemics of the past century (see References: WHO: Avian influenza: assessing the pandemic threat). These lessons are summarized as follows.

• Pandemics behave as unpredictably as the viruses that cause them. During the previous century, great variations were seen in mortality, severity of illness, and patterns of spread.
• One consistent feature important for pandemic preparedness planning is the rapid surge in the number of cases and their exponential increase over a very brief time, often measured in weeks.
• Apart from the inherent lethality of the virus, its capacity to cause severe disease in non-traditional age groups, namely young adults, is a major determinant of a pandemic's overall impact.
• The epidemiologic potential of a virus tends to unfold in waves. Subsequent waves have tended to be more severe.
• Virologic surveillance, as conducted by the WHO Laboratory Network, has performed a vital function in rapidly confirming the onset of pandemics.
• Most pandemics have originated in parts of Asia where dense populations of humans live in close proximity to ducks and pigs.
• Some public health interventions may have delayed the international spread of past pandemics, but could not stop them.
• Delaying spread is desirable, as it can flatten the epidemiological peak, thus distributing cases over a longer period of time.
• The impact of vaccines on a pandemic, through potentially great, remains to be demonstrated. In 1957 and 1968, vaccine manufacturers responded rapidly, but limited production capacity resulted in the arrival of inadequate quantities too late to have an impact.
• Countries with domestic manufacturing capacity will be the first to receive vaccines.
• The tendency of pandemics to be most severe in later waves may extend the time before large supplies of vaccine are needed to prevent severe disease in high-risk populations.
• In the best-case scenario, a pandemic will cause excess mortality at the extremes of the lifespan and in persons with underlying chronic disease. Countries with good programs for yearly influenza vaccinations will have experience with the logistics of vaccinations for these populations.

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The Pandemic Severity Index

In February 2007, HHS released the "pandemic severity index," or PSI, as a way to grade pandemics. The severity level is initially based on case-fatality ratio (CFR), a single criterion that will likely be known even early in a pandemic when small clusters and outbreaks are occurring. The pandemic severity index levels are:

• Category 1, CFR <0.1%
• Category 2, CFR 0.1% to 0.5%
• Category 3, CFR 0.5% to 1%
• Category 4, CFR 1% to 2%
• Category 5, CFR >2%

According to this index, the pandemics of 1957 and 1968 both fit into Category 2, whereas the 1918 pandemic qualified as a Category 5.

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The Current H5N1 Threat

According to WHO, at this time the pandemic alert level for H5N1 influenza is at Phase 3: a new viral subtype is causing disease in humans but is not yet spreading efficiently and sustainably (see References: WHO: Current WHO phase of pandemic alert).

Detailed information about H5N1 influenza in bird populations can be found in the document on this Web site "Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations" and in human populations in the document "Avian Influenza (Bird Flu): Implications for Human Disease."

Of the avian influenza subtypes, currently the H5N1 subtype is of greatest pandemic concern for the following reasons (see References: WHO: Avian influenza fact sheet; WHO: Influenza pandemic preparedness and response):

• H5N1 viruses have spread rapidly throughout poultry flocks in Asia over the past 2 years and now appear to be endemic in eastern Asia (see References: Kaye 2005, Li 2004). In addition, H5N1 viruses have spread beyond Asia via migratory birds to several countries in Europe and Africa and to India.
• H5N1 strains cause severe disease in humans, with a high case-fatality rate (reportedly at over 60%, although adequate surveillance data are lacking to accurately define the rate).
• The potential of exposure and infection of humans is likely to be ongoing in rural Asia and probably in Africa as well, where many households keep free-ranging poultry flocks for income and food (see References: Stohr 2005).
• The viruses have diversified genetically into two distinct phylogenetic clades (genetic groups) and subclades, indicating ongoing viral evolution (see References: European Centre for Disease Surveillance and Control 2006; WHO 2007: Antigenic and genetic characteristics of H5N1 viruses and candidate H5N1 vaccine viruses developed for potential use as pre-pandemic vaccines).
• Clade 1 viruses have circulated in Cambodia, Thailand, and Vietnam, and clade 2 viruses have circulated in China and Indonesia and have spread westward to the Middle East, Europe, and Africa.
• Six different subclades of clade 2 have been recognized and three of these have been responsible for most of the human cases in Indonesia, China, and outside of Asia.
• Recent genetic sequencing performed on H5N1 viral isolates from Turkey demonstrates that the strains contain two mutations which may make the virus better adapted to humans (see References: Butler 2006). These mutations could potentially enhance transmission from birds to humans and between humans.

If H5N1 strains continue to circulate widely among poultry, the potential for emergence of a pandemic strain remains high. For example, H5N1 viruses have been found in pigs in southern China, and human H3N2 influenza viruses are endemic in pigs in that area. H5N1 has recently been reported in pigs in Indonesia as well (see References: Cyranoski 2005). Thus, the conditions exist for exchange of genetic material between the different viruses in the pig host (see References: Li 2004; WHO: Avian influenza: update: implications of H5N1 infections in pigs in China). Some scientists believe that reassortment between an avian and a human strain could occur in the human population without an intermediary host; if this proves true, as more humans become exposed and infected, the potential for reassortment with a human strain also may increase. Similarly, as more human cases occur globally and the virus gains a foothold in the human population, the potential for gradual adaptation of the virus into a human pandemic strain increases (see References: WHO: Influenza pandemic preparedness and response 2005).

Since 2003, human cases of H5N1 influenza have been reported in Azerbaijan, Cambodia, China, Djibouti, Egypt, Indonesia, Iraq, Lao People's Democratic Republic, Myanmar, Nigeria, Pakistan, Thailand, Turkey, and Vietnam.

• WHO has officially recognized more than 380 cases of H5N1 influenza (see References: WHO: Cumulative number of confirmed human cases of avian influenza), with an overall case-fatality rate of more than 60%. The reported case-fatality rate among cases in Indonesia is higher, at over 80%.
• An epidemiologic report on 203 confirmed H5N1 influenza cases published by WHO in June 2006 demonstrated that the median age of cases was 20 years and that 90% of infections occurred in persons under 40 years of age (see References: WHO: Epidemiology of WHO-confirmed human cases of avian A(H5N1) infection).

The high case-fatality rate suggests that the pathogenicity of H5N1 may be similar to (or more severe than) the 1918 H1N1 pandemic strain. Researchers have hypothesized that cytokine storm (ie, overproduction of cytokines) may have played an important role in the pathogenesis of the 1918 pandemic strain. A laboratory-based study involving H5N1 strains taken from ill humans in Asia (during 1997 and 2004) and an ordinary current H1N1 strain (circulating in Asia in 1998) found that all the H5N1 viruses caused human alveolar cells and bronchial epithelial cells to secrete significantly higher levels of various cytokines and chemokines than did the ordinary virus (see References: Chan 2005). Another recent study demonstrated a strong induction of chemokines and their receptors in macrophages infected by H5N1 and H9N2 avian influenza viruses (see References: Zhou 2006). Finally, a recent case series reported from Vietnam involving patients with H5N1 influenza showed that high viral load and high chemokine and cytokine levels are central to the pathogenesis of H5N1 influenza (see References: de Jong 2006). These findings support the role of cytokine storm in the pathogenesis of H5N1.

To date, sustained person-to-person transmission has not been recognized, although probable person-to-person spread was identified in Thailand involving transmission from an ill child to her mother and aunt (see References: Ungchusak 2005) and several other familial clusters have been recognized (see References: Olsen 2005). In May 2006, WHO reported an H5N1 influenza cluster in Indonesia involving seven cases of person-to-person transmission; one of the cases involved two generations of transmission (see References: WHO: Avian influenza: situation in Indonesia: update 14, and see May 24, 2006, CIRAP News story). An Indosesian official recently put the number of clusters in that country at 10, all involving cases in blood relatives (see Jan 12, 2007, CIDRAP News story). Inefficient transmission of current H5N1 strains may be related to lack of appropriate avian virus cell receptors in the upper respiratory tracts of humans and the inability of H5N1 strains to recognize human cell receptors (see References: Shinya 2006). A mutation allowing H5N1 avian influenza virus to recognize human cell receptors could enhance person-to-person transmission owing to the potential for greater viral replication in the upper respiratory tract.

Public health officials are concerned about the continuing evolution and spread of H5N1 (see References: Webster 2006). They are closely monitoring the situation and watching for the emergence of a strain capable of causing sustained human-to-human transmission.

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Vaccine Development

Development of an effective vaccine is considered the cornerstone for controlling a global influenza pandemic. In general, if a novel strain occurs without adequate warning, WHO has indicated that it will take at least 4 to 6 months to develop a vaccine (see References: WHO: WHO global influenza preparedness plan 2005). However, there are several major obstacles in producing an adequate vaccine supply during a pandemic:

• Limited production capacity
• Production capability in only a few countries, which will yield an inequitable distribution
• Technological challenges to vaccine development

Limited Production Capacity

For the period 2000 to 2003, global annual influenza vaccine production ranged from approximately 230 million doses of trivalent vaccine (2000) to 291 million doses (see References: Fedson 2004: Pandemic influenza vaccine: obstacles and opportunities; Medema 2004).

• In the "best case scenario," assuming that the pandemic vaccine would be a single-dose monovalent vaccine requiring the same level of antigen per dose (15 mcg HA), the production capacity would be increased to an estimated 750 million doses each year (see References: WHO: Consultation on priority public health interventions before and during an influenza pandemic).
• However, current egg-based production methods do not work nearly as well for production of H5N1 flu vaccines as they do for seasonal flu vaccines. As a result, WHO estimates that the current maximum capacity for H5N1 vaccine is about 500 million (single-strain) doses each year. If a two-dose schedule is required, only 250 million vaccine courses would be available each year (see References: WHO: Global pandemic influenza action plan to increase vaccine supply).
• In the United States, domestic production was estimated at 50 million doses of trivalent vaccine during 2004. This would be equivalent to about 150 million doses of monovalent standard-dose, assuming 15 mcg HA per dose (see References: Fedson 2003).
• Two critical caveats need to be considered with these types of estimates: (1) it is not clear how many micrograms of antigen will be necessary to elicit an immune response to a pandemic strain (it may be that 30 to 90 mcg per dose may be needed to illicit an adequate immune response) and (2) two doses of vaccine will likely be needed to confer adequate protection. A vaccine requiring two doses and 90 mcg per dose would provide enough vaccine for only 75 million people worldwide, given the current vaccine production capacity (see References: Poland 2006).

Production Capability in Only a Few Countries

Most of the world's influenza vaccine is produced in a few countries. These countries are likely to reserve scarce supplies for their own populations during a pandemic, thus leading to an inequitable distribution of vaccine, particularly to developing countries. This issue has relevance for the United States as well, where current domestic vaccine production falls far short of producing adequate vaccine supplies to vaccinate the entire US population. Moreover, the US plan does not address the issue of distributing vaccine to other countries.

Nine companies, located in the following nine developed countries, currently produce influenza vaccine:

• Australia
• Canada
• France
• Germany
• Italy
• The Netherlands
• Switzerland
• The United Kingdom
• The United States

Technological Challenges to Vaccine Development

The manufacture of vaccines derived from pathogenic avian strains poses a number of technological challenges. For example, highly pathogenic avian strains cannot be grown in large quantities in eggs because they are lethal to chick embryos. These strains also pose significant safety issues and would require extensive biocontainment procedures during the manufacturing process.

Several approaches have been suggested to overcome these issues. One approach, use of reverse genetics, has been used for preparing H5N1 seed strains (see References: Webby 2004; WHO: Development of a vaccine effective against avian influenza H5N1 infection in humans). Reverse genetics provides several advantages in influenza vaccine development (see References: Luke 2006, Palese 2006): (1) it allows creation of engineered viruses that are modified to be less virulent, thus eliminating the need for high-level containment, (2) with reverse genetics, a selection system is not needed to derive appropriate reassortant viruses from background parental viruses, (3) it dramatically shortens the timeframe for production of seed strains, (4) it allows for standardization of seed strains to be used in vaccine development, and (5) the process may eliminate the potential for any adventitious agents to enter the manufacturing process. Other approaches include the following (see References: Stephenson 2004).

• Produce inactivated vaccine from wild-type virus
• Select an antigenically related but nonpathogenic surrogate vaccine strain
• Use other viruses (eg, baculoviruses, adenoviruses) to express recombinant hemagglutinin
• Develop DNA-based vaccines

It is not yet clear whether new vaccines made from seed strains generated through reverse genetics will be immunogenic in humans, given that candidate vaccines developed against the 1997 H5N1 strain from Hong Kong were poorly immunogenic (see References: Stephenson 2004). It may be that an effective vaccine cannot be developed until a true pandemic strain (reassorted with human influenza viruses) emerges and can be used as the seed virus.

In May 2006, HHS awarded $1 billion to five pharmaceutical companies to develop cell-based technologies for making influenza vaccines. The vaccine manufacturers are GlaxoSmithKline, MedImmune, Novartis Vaccines & Diagnostics, DynPort Vaccine, and Solvay Pharmaceuticals (see May 4, 2006, CIDRAP News story).

Research into new approaches for vaccine production is a high priority because stockpiling prototype vaccines may be worthwhile if protection against emergent strains can be demonstrated (see References: Schwartz 2005). Recent studies using prototype vaccines developed through reverse genetics or recombinant technology suggest that these strategies are promising:

• One study demonstrated good cross-protection against H5N1 in mice following vaccination with an H5 influenza vaccine created through reverse genetics (see References: Lipotov 2005). Protection was achieved despite antigenic differences and incomplete matching between the vaccine strain and the challenge virus. Although these findings are promising, it is not clear if similar protection would occur for humans.
• A second recent study found that an inactivated whole-virus H5N1 vaccine produced through reverse genetics offered protection to ferrets challenged with the vaccine strain and to ferrets challenged with two other H5N1 strains (see References: Govorkova 2006).
• Two additional studies have tested the immunogenicity of recombinant adenovirus-based H5N1 vaccines. One study demonstrated protection against lethal challenge in mice (see References: Hoelscher 2006) and the other demonstrated protection in mice and chickens (see References: Gao 2006).

Another option for consideration is development of influenza vaccines based on cell-mediated immunity. Cell-mediated responses generally focus on internal influenza proteins, which are more conserved and less susceptible to antigenic variation (see References: Thomas 2006).

Interpandemic Steps to Facilitate Vaccine Production

In September 2006, WHO released an action plan to increase pandemic influenza vaccine production capacity (see References: WHO: Global pandemic influenza action plan to increase vaccine supply). The plan outlines the following strategies:

• Develop an immunization policy to increase demand for seasonal vaccines.
• Develop regional and national plans for seasonal influenza vaccination programs.
• Mobilize resources for the implementation of seasonal influenza vaccination programs.
• Increase influenza vaccine production capacity.
• Increase capacity for inactivated influenza vaccines.
• Improve production yield of H5N1 viruses and immunogenicity of prototype H5N1 inactivated vaccine.
• Build new production facilities in developing and/or industrialized countries.
• Assess formulations of influenza vaccine other than those commonly used for seasonal vaccine.
• Conduct clinical trials of adjuvanted vaccines.
• Explore the possibility to scale-up production of live, attenuated influenza vaccines.
• Further evaluate whole-cell vaccines.
• Assess alternative vaccine delivery routes (such as intradermal administration).
• Promote research and development for new influenza vaccines.
• Enhance protective efficacy and immunogenicity of existing vaccine types.
• Develop novel vaccines that induce broad-spectrum and long-lasting immune responses.
• Improve evaluation of vaccine performance.

Current Status of H5N1 Candidate Vaccines

Because of concerns about the pandemic potential of H5N1, WHO has been working with laboratories in its influenza network to develop vaccines against this subtype (see References: WHO: Development of a vaccine effective against avian influenza H5N1).

• Candidate vaccines were developed during 2003 by network laboratories in London and in Memphis, Tennessee, for protection against the strain that was isolated from humans in Hong Kong in February of that year. However, the 2004 strain is different from that strain.
• In April 2004, WHO made the prototype seed strain for an H5N1 vaccine available to manufacturers (see References: WHO: Avian influenza: situation in Thailand; status of pandemic vaccine development). In August 2006, WHO changed the prototype strains and now offers three new prototype strains which represent three of the six subclades of the clade 2 virus; these strains have been responsible for many of the human cases that have occurred since 2005 (see References: WHO: Antigenic and genetic characteristics of H5N1 viruses and candidate H5N1 vaccine viruses developed for potential use as pre-pandemic vaccines and see Aug 18, 2006, CIDRAP News Story).
• The National Institute of Allergy and Infectious Diseases (NIAID) awarded two contracts to support the production and clinical testing of an investigational vaccine based on the prototype seed strain made available by WHO (see References: NIAID 2004). The contracts were awarded to Aventis Pasteur (now Sanofi Pasteur) of Swiftwater, Pennsylvania, and to Chiron Corporation of Emeryville, California. Each manufacturer is using established techniques in which the virus is grown in eggs and then inactivated and further purified before being formulated into vaccines.
• A recently published report involving a Sanofi Pasteur H5N1 vaccine found that only 54% of 99 subjects who received two doses of vaccine (90 mcg of HA per dose) had neutralization antibody titers that reached 1:40 or greater (see References: Treanor 2006).
• Another report involving two doses of a 30-mcg H5N1 Sanofi Pasteur vaccine with alum added (an adjuvant used in many vaccines to boost immune response) found that vaccination elicited an immune response in 67% in 51 volunteers (see References: Bresson 2006).
• In July 2006, GlaxoSmithKline (GSK) released preliminary information on a clinical trial using an adjuvanted H5N1 influenza vaccine (see Jul 26, 2006, CIDRAP News story). Results showed that 80% of volunteers who received two vaccine doses containing at least 3.8 mcg of antigen with an adjuvant had a strong immune response (ie, a hemagglutination inhibition titer of 40). The company tested the vaccine on 400 adult Belgians between the ages of 18 and 60, using four different antigen doses, with 3.8 mcg the lowest. The GSK vaccine was made from an inactivated H5N1 virus collected in Vietnam in 2004.
• A phase 1 randomized trial of an inactivated adjuvanted whole-virion H5N1 vaccine showed that 78% of volunteers developed an antibody response after 2 doses of 10 mcg hemagglutinin per dose; the adjuvant used was aluminum hydroxide (see References: Lin 2006).
• The intramural research program of NIAID also has generated live, attenuated, cold-adapted H5N1 and H9N2 vaccine candidates that have been protective in mice (see References: Fauci 2006). Further work on development of live, attenuated pandemic vaccines is ongoing (see References: Luke 2006).
• Researchers have suggested that development and use of an H5N1 vaccine for immunologic priming during the interpandemic period may offset the need for two doses of vaccine once a pandemic begins (assuming the pandemic is caused by H5N1), even if the strain used in the priming vaccine is somewhat different from an emergent pandemic strain (see References: Monto 2006).
• One study demonstrated varying degrees of cross-reactivity to H5N1 strains in 14 subjects following vaccination with MF59-adjuvanted H5N3 vaccine, suggesting that vaccines made from other H5 strains could be used as part of a priming strategy (see References: Stephenson 2005).
• In the late 1990s, a group of 37 individuals received two doses of an experimental Hong Kong H5N1 vaccine (developed from a clade 3 virus). The group recently was revaccinated as part of a clinical trial with a different H5N1 vaccine from a clade 1 strain from Vietnam. Investigators found that more than twice as many of the individuals who had received the priming dose of clade 3 H5N1 vaccine responded with substantial antibody levels to a single dose of the clade 1 H5N1 vaccine than did those with no prior H5N1 exposure. These findings suggest that priming with an H5N1 vaccine before a pandemic may be a useful strategy (see CIDRAP News story, Oct 13, 2006).
• A cell-based vaccine produced by Baxter (using vero cell–based technology, inactivated whole virus of H5N1 avian influenza strain A/Vietnam/1203/2004, and aluminum oxide adjuvant) found that the vaccine was relatively immunogenic at low doses (3.75 and 7.5 mcg) and also showed evidence of cross-protection against other H5N1 strains (see Oct 17, 2006, CIDRAP News story).
• A universal influenza vaccine could provide protection against all types of influenza and would eliminate the need to develop individual vaccines to specific H and N virus types (see References: Gerhard 2006). ch a vaccine would not need to be reengineered each year and could protect against an emergent pandemic strain. Developing a universal vaccine requires that researchers identify conserved regions of the influenza virus that do not exhibit antigenic variability by strain or over time. A universal vaccine is being developed by the British company Acambis and is being researched by others as well. Acambis announced in early August 2005 that it has had successful results in animal testing (see References: Acambis 2005). The vaccine focuses on the M2 viral protein, which does not change, rather than the surface hemagglutinin and neuraminidase proteins targeted by traditional flu vaccines. The universal vaccine is made through bacterial fermentation technology, which would greatly speed up the rate of production over that possible with culture in chicken eggs, plus the vaccine could be produced constantly, since its formulation would not change. Still, such a vaccine is years away from full testing, approval, and use.
• WHO indicated in a statement released on February 16, 2007, that progress is being made on development of prototype pandemic influenza vaccines (see References: WHO reports some promising results on avian influenza vaccines). Sixteen manufacturers from 10 countries are developing prototype pandemic influenza vaccines against H5N1 avian influenza virus. Five of them also are involved in the development of vaccines against other avian viruses (H9N2, H5N2, and H5N3). More then 40 clinical trials involving prototype vaccines have been completed or are ongoing. Even thought these findings are encouraging, WHO also expressed concern about global vaccine production capacity.

Development of Vaccines Against Influenza Viruses Other Than H5N1 That May Pose a Pandemic Threat

• In mid-September 2006, Sanofi Pasteur announced that they are beginning a clinical trial of an H7N1 vaccine; the vaccine is a split virus product grown in cell culture rather than in eggs (see Sep 19, 2006, CIDRAP News story).
• As noted in the section above, live, attenuated, cold-adapted H9N2 vaccine candidates have been developed that are protective in mice (see References: Fauci 2006).
• A phase 1, randomized, double-blind trial of an H9N2 subunit vaccine with and without MF59 adjuvant showed that the adjuvanted vaccine was immunogenic even after a single dose (see References: Atmar 2006).

Stockpiling H5N1 Vaccines and Vaccination Strategies

Currently, the US government has a stockpile of 5.9 million doses of H5N1 vaccine. While the government has stockpiled approximately 7.5 million doses, officials estimate that about 1.4 million of those doses have begun to lose potency and another 200,000 have been used in research (see Nov 17, 2006, CIDRAP News Story).

In November 2006, HHS announced the awarding of three contracts to buy an additional 5.3 million 90-mcg doses of H5N1 vaccine (see Nov 20, 2006, CIDRAP News Story). HHS hopes eventually to have enough H5N1 vaccine stockpiled to vaccinate 20 million people.

In contrast to the US government’s approach to stockpiling H5N1 vaccine, WHO recently released a document cautioning world governments that too many scientific uncertainties need to be addressed before global recommendations can be made in favor of stockpiling H5N1