Avian Influenza (Bird Flu): Implications for Human Disease

Last updated August 28, 2008

Agent
Laboratory Testing for Avian Influenza in Humans
Summary of Avian Influenza in Humans
The Current Outbreak of H5N1 in Birds and Other Animals
H5N1 in Humans: Epidemiologic Features
H5N1 in Humans: Clinical Features
Treatment and Prophylaxis
Current Status of H5N1 Candidate Vaccines
Current WHO and CDC Travel Recommendations
Use of Seasonal Flu Vaccine in Humans at Risk for H5N1 Infection
Surveillance Considerations
Influenza Pandemic Considerations
Infection Control Recommendations
Guidance to Protect Workers from Avian Influenza Viruses
Food Safety Issues
References

Agent

Avian influenza is caused by influenza A viruses. More information about avian influenza in bird populations can be found in the document "Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations" on this site.

Family: Orthomyxoviridae

Enveloped virions are 80 to 120 nm in diameter and 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.
All past influenza pandemics have been caused by influenza A viruses.
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: 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).
All known subtypes of influenza A can be found in birds, and wild aquatic birds are the major reservoir for influenza A viruses (see References: Fouchier 2004).
Human disease historically has been caused by three subtypes of HA (H1, H2, and H3) and two subtypes of NA (N1 and N2). H1 and H3 are the subtypes that currently cause seasonal influenza in human populations around the globe each year.
More recently, human disease has been recognized to be caused by additional HA subtypes, including H5, H7, and H9. Such cases have predominantly been associated with exposure to infected birds. Person-to-person transmission has occurred in a few isolated situations.

Other mammalian hosts for influenza A

Influenza A viruses have traditionally been known to cause disease in horses, pigs, whales, and seals.
H5N1 influenza A has now been shown to infect cats, leopards, tigers, civets, and possibly dogs (see References: European Centre for Disease Prevention and Control Influenza Team 2006: H5N1 infections in cats; Keawcharoen 2004; Kuiken 2004; Songserm 2006; Thanawongnuwech 2005; Webster 2006; Yingst 2006; and see Aug 31, 2006, CIDRAP News story). Asymptomatic infection has been reported in domestic cats (see References: Leschnik 2007).
H5N1 recently was isolated from an infected mink and a stone marten in Europe (see References: WHO 2006: Influenza research at the human and animal interface).
A recent report involving cats experimentally infected with H5N1 demonstrated that infected cats excreted the virus via the respiratory tract and the digestive tract, suggesting that in addition to the respiratory route, other routes of transmission may play a role in spread among mammalian hosts (see References: Rimmelzwaan 2006). Cat-to-cat transmission of H5N1 can occur (see References: WHO 2006: Influenza research at the human and animal interface). Some experts are concerned that cats could play a role in transmission of H5N1 to humans, although this has not been documented to date (see References: Kuiken 2006). Based on these considerations, FAO recommends that avian influenza in cats should be closely monitored (see References: FAO: 2007: Avian influenza in cats should be closely monitored).
H5N1 was identified in pigs in China in 2001 and 2003 (see References: Cyranoski 2004). The virus also was found in pigs in Indonesia in 2005 when 5 of 10 pigs tested in western Java were shown to be asymptomatically infected (see References: Cyranoski 2005) and again in 2006 on the Indonesian island of Bali (see Oct 10, 2006, CIDRAP New story). A recent laboratory study found that domestic pigs have low susceptibility to H5N1 viruses; experimental inoculation resulted in asymptomatic infection or mild symptomatic infection limited to the respiratory tract and tonsils (see References: Lipatov 2008).
A recent report demonstrated that calves can be experimentally infected with H5N1 virus (see References: Kalthoff 2008).

Cases of canine influenza caused by H3N8 recently have been recognized in the United States; this subtype traditionally has been found in horses (see References: Crawford 2005, Yoon 2005).

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.
Severe disease from influenza generally does not develop in wild birds; however, recently H5N1 has been shown to be virulent for wild bird species. An outbreak of H5N1 among migratory geese and other wild birds in Qinghai province, China, was identified in May 2005 (see References: Chen 2005, Liu 2005). An outbreak in wild swans occurred in Azerbaijan in February 2006, and severe illness from H5N1 influenza has been recognized in a variety of other wild bird species (see References: Gilsdorf 2006; Olsen 2006; USGS National Wildlife Health Center).
Outbreaks of influenza have been recognized in domestic poultry (chickens and turkeys) for many years. 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. All HPAI strains identified to date have involved H5 and H7 subtypes.
Human infections caused by avian strains have been associated with both HPAI and LPAI strains (H5, H7, and H9) (see References: HHS 2005: Pandemic influenza plan).
Evidence that HPAI strains arise from LPAI strains has led the World Organization for Animal Health (OIE) 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). The pandemic strains of 1957-58 (H2N2) and 1968-69 (H3N2) both involved reassortment events between avian and human influenza strains.

H5 subtypes

H5 subtypes can be found throughout the world and include both LPAI and HPAI strains.
H5N1 is responsible for the current panzootic among domestic poultry and other birds in Asia, the Middle East, Europe, and Africa.
Recent genetic characterization of H5N1 strains involved in the current panzootic has demonstrated two distinct phylogenetic clades (clades 1 and 2) (see References: Webster 2006; WHO Global Influenza Program Surveillance Network; WHO: Antigenic and genetic characteristics of H5N1 viruses and candidate H5N1 vaccine viruses developed for potential use as human vaccines). Six different subclades of clade 2 have been recognized; three of these are primarily responsible for recent human H5N1 cases.

Clade 1 viruses have circulated primarily in Cambodia, Thailand, Hong Kong, and Vietnam.
Clade 2.1 viruses have circulated primarily in Indonesia.
Clade 2.2 viruses have a wide geographic distribution and have spread to over 60 countries in Asia, the Middle East, Europe, and Africa.
Clade 2.3 viruses are genetically diverse and continue to circulate in birds in Asia. Viruses from this group have caused illness in humans in China, Lao People's Democratic Republic (PDR), Myanmar, and Vietnam.

H7 and H9 subtypes

H7 includes HPAI and LPAI strains.
H9 is only known to include LPAI strains. H9N2 viruses had been isolated in multiple avian species throughout Asia, the Middle East, Europe, and Africa.
These subtypes have caused infections in humans on rare occasions (see References: CDC: Avian influenza A viruses; NIAID: Timeline of human pandemics). A recent report, however, suggests that human infections with H9N2 viruses may be more common than previously recognized (see References: Wan 2008). The authors also concluded that H9N2 viruses can evolve extensively and reassort, suggesting that they may be capable of undergoing further adaptation for more efficient transmission among mammals, including humans.

Influenza A nomenclature

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 (for human strains) include: A/Hong Kong/03/68[H3N2], A/swine/Iowa/15/30[H1N1]).
As with other influenza A subtypes, standard nomenclature is used to name avian strains (eg, A/chicken/HK/5/98 [H5N1]).

Environmental survival of avian influenza viruses

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.)
Influenza A viruses can persist for extended periods in water (see References: WHO: Review of latest available evidence on risks to human health through potential transmission of avian influenza [H5N1] through water and sewage). One study of subtype H3N6 found that virus resuspended in Mississippi River water was detected for up to 32 days at 4°C and was undetectable after 4 days at 22°C (see References: Webster 1978). Another study found that several avian influenza viruses persisted in distilled water for 207 days at 17°C and 102 days at 28°C (see References: Stallknecht 1990).
Influenza A viruses can be preserved in lake ice and then released when the ice thaws the following spring or, in the case of arctic ice, up to years later. This may lead to temporal gene flow between viruses entrapped during one year and those shed by migrating birds in following years (see References: Zhang 2006).
Recent data from studies of H5N1 in domestic ducks have shown that H5N1 can survive in the environment for 6 days at 37ºC (see References: WHO: Laboratory study of H5N1 viruses in domestic ducks).
Inactivation of the virus occurs under the following conditions (see References: OIE 2002, PHS):

Temperatures of 56°C for 3 hours or 60°C or more for 30 minutes
Acidic pH conditions
Presence of oxidizing agents such as sodium dodecyl sulfate, lipid solvents, and B-propiolactone
Exposure to disinfectants: formalin, iodine compounds

Laboratory Testing for Avian Influenza in Humans

General Considerations

Tests for influenza include: viral culture, polymerase chain reaction (PCR), rapid antigen testing, and immunofluorescence.
Laboratory tests are widely used to identify influenza virus at the genus level (influenza A/B) or at the H-type level (H1, H3, and H5).
H subtype-specific tests must be used to identify potential avian strains, including H5N1.
The World Health Organization (WHO) recommends forwarding all H5, H7, and H9-positive isolates to an approved influenza reference laboratory for confirmation and N-typing (see References: WHO: Guidelines for global surveillance of influenza A/H5; WHO: Recommendations and laboratory procedures for detection of avian influenza A [H5N1] virus in specimens from suspected human cases).
Serologic tests have been used to diagnose infection retrospectively.
During a pandemic alert period for an avian influenza virus, 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.
During a pandemic (involving an avian strain or other strain), recommendations for laboratory testing may be unique and depend on factors such as: (1) availability of reagents and laboratory surge capacity, (2) 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 avian influenza and other emerging influenza variants (see References: Weinberg 2005).
Laboratory-based influenza surveillance networks

WHO Global Influenza Surveillance Network (see References)
Centers for Disease Control and Prevention (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). Information also was taken from the Department of Health and Human Services (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

Plasma in EDTA (for detection of viral RNA)
Rectal swab (for patients with diarrhea)
Spinal fluid (if meningitis is suspected 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 if 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 of 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 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 8oC) 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

Biosafety and Biosecurity

Biosafety

Updated safety rules and recommendations for influenza virus have been included in the fifth edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL) (see References: HHS: Biosafety in Microbiological and Biomedical Laboratories [BMBL] 5th Edition). Current recommendations for interpandemic and pandemic alert periods include:

Biosafety level 2 (BSL-2) facilities, practices, and procedures are recommended for diagnostic, research, and production activities utilizing contemporary, circulating human influenza strains (eg, H1/H3/B), LPAI strains (eg, H1-4, H6, H8-16) and equine and swine influenza viruses.

Animal biosafety level 2 (ABSL-2) facilities are appropriate for work with these viruses in animal models.
Use of avian and swine influenza viruses requires a permit from the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture (USDA).
Based on economic ramifications and source of the virus, LPAI H5 and H7 and swine influenza viruses may have additional APHIS permit-driven containment requirements, personnel practices, and/or restrictions.

Noncontemporary, wild-type human influenza (H2N2) strains should be handled with increased caution. Important considerations in working with these strains are the number of years since an antigenically related virus last circulated and the potential for a susceptible population.

BSL-3 and ABSL-3 practices, procedures, and facilities are recommended with rigorous adherence to additional respiratory protection and clothing change protocols.
Negative-pressure, high-efficiency particulate air (HEPA)-filtered respirators, or positive air-purifying respirators (PAPRs) are recommended for use.
Cold-adapted, live attenuated H2N2 vaccine strains may continue to be worked with at BSL-2 facilities.

Any research involving reverse genetics of the 1918 influenza strain should proceed with extreme caution. The risk to laboratory workers is unknown at present, but the pandemic potential is thought to be significant. Until further risk assessment data are available, the following practices and conditions are recommended for manipulation of reconstructed 1918 influenza viruses and laboratory animals infected with the viruses. These practices and procedures are considered minimum standards for work with the fully reconstructed virus.

BSL-3 and ABSL-3 practices, procedures, and facilities
Large laboratory animals, such as nonhuman primates, housed in primary-barrier systems in ABSL-3 facilities
Rigorous adherence to additional respiratory protection and clothing change protocols
Use of negative-pressure, HEPA-filtered respirators or PAPRs
Use of HEPA filtration for treatment of exhaust air
Amendment of personnel practices to include personal showers prior to exiting the laboratory

Manipulating HPAI viruses in biomedical research laboratories requires similar caution, because some strains may pose increased risk to laboratory workers and have significant agricultural and economic implications.

BSL-3 and ABSL-3 practices, procedures, and facilities are recommended, along with clothing change and personal showering protocols.
Loose-housed animals infected with HPAI strains must be contained within agriculture-specific BSL-3 (BSL-3-Ag) facilities.
Negative-pressure, HEPA-filtered respirators or PAPRs are recommended for HPAI viruses with potential to infect humans.

When considering the biocontainment level and attendant practices and procedures for work with other influenza recombinant or reassortant viruses, the local Institutional Biosafety Committee should consider, but not limit consideration, to the following in the conduct of protocol-driven risk assessment.

The gene constellation used
Clear evidence of reduced virus replication in the respiratory tract of appropriate animal models, compared with the level of replication of the wild-type parent virus from which it was derived
Evidence of clonal purity and phenotypic stability
The number of years since a virus that was antigenically related to the donor of the HA and NA genes last circulated
If adequate risk assessment data are not available, a more cautious approach utilizing elevated biocontainment levels and practices is warranted. There may be specific requirements regarding the setting of containment levels in institutions that are subject to NIH guidelines.

Recommendations for testing of clinical specimens from patients suspected to have H5N1 influenza include (see References: CDC: Update on influenza A [H5N1] and SARS):

Culture from patients suspected of having avian influenza, other novel influenza strains, or severe acute respiratory syndrome (SARS) coronavirus should be conducted only 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, probable H1 or H3 isolates) to prevent recombination.
Indirect immunofluorescence (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 reverse transcriptase polymerase chain reaction (RT-PCR), should be conducted under BSL-2 conditions 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 as described for H5N1 avian influenza.
Additional requirements and recommendations apply for laboratory work involving live animals.

Biosecurity

Strains of HPAI and the 1918 influenza virus are Select Agents requiring registration with CDC and/or USDA for possession, use, storage, and/or transfer (see References: HHS: Biosafety in Microbiological and Biomedical Laboratories [BMBL] 5th Edition).
HPAI strains are agricultural Select Agents requiring registration of personnel and facilities with the lead agency for the institution (CDC or USDA-APHIS) (see References: USDA/APHIS: Agricultural Bioterrorism Protection Act of 2002). An APHIS permit is required for working with these agents. Additional containment requirements, personnel practices, and/or restrictions may be added as conditions of the permit.
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).

Direct Detection Methods

RT-PCR assays

RT-PCR assays use conserved targets such as the matrix (M) protein for genus-level identification. HA and NA targets are used for specific identification of avian subtypes. PCR generally is not used for strain-level identification, which is based on serologic markers.
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).
In February 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 the CDC to state and city LRN (Laboratory Response Network) laboratories. Testing with the new assay is limited to LRN-designated laboratories.
Multiplex real-time RT-PCR assays have been developed for specific detection of virus subtype H5 or H5N1 (See References: Kessler 2004, Ng 2005, Payungporn 2005).

One recent study evaluated an influenza virus subtype H5 RT-PCR assay based on a TaqMan-minor groove binder (MGB) probe (see References: Lu 2008). The assay took less than 3 hours to complete and showed good sensitivity and repeatability.
Another recent study described a multiplex RT-PCR assay for simultaneous detection of influenza virus types A and B and subtypes H5 and N1 (see References: Wu 2008).

Multiplexed RT-PCR assays have been developed that can detect and identify 12 HA (H1 through H12) and 9 NA (N1 through N9) subtypes commonly isolated from birds, pigs, and humans (see References: Chang 2008).

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 2005: Pandemic influenza plan).
Common PCR targets include the M protein (for genus-level identification) and HA and NA (for subtype-level identification). PCR generally is not used for strain-level identification, which is based on serologic markers.
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 the CDC for confirmation (see References: HHS 2005: 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 2005: 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—HA, NA, and 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; Treanor 2005; WHO: Checklist for influenza pandemic preparedness planning)

Rapid tests detect viral antigen (generally nucleoprotein) or enzymatic activity (NA) 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; therefore, positive results should be confirmed by culture or RT-PCR. When the disease prevalence is high, the negative predictive value of the tests will be lower and clinicians should consider confirming negative tests with culture or RT-PCR.
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).
The WHO, in its 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).

Serology

Serologic testing can be used for retrospective diagnosis of infection but is rarely useful for patient management and is not widely available (see References: Hayden 2002; Treanor 2005; HHS 2005: 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), hemagglutination inhibition (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).
Immunoglobulin type G (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 for 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 2005: Pandemic influenza plan).
HAI and EIAs measure antibody to HA. 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 2005: 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 2005: Pandemic influenza plan).

Virus Isolation by Cell Culture

Virus isolation is considered the "gold standard" of influenza testing (see References: Hayden 2002, Treanor 2005).
Culture of specimens from suspect cases of avian influenza requires special containment facilities, procedures, and registration (see above). Samples from cases without specific risk factors may be cultured using standard facilities and procedures.
Unlike antigen or nucleic acid–based tests, a positive result is considered definitive for the diagnosis.
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 2005: 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:

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 and mumps viruses, may also cause HAd. The lack of HAd specificity may be an advantage in detecting new or pandemic strains.
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 HAI 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), 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 an 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.

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 or suspected to be responsible for resistance
NA 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 NA 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).

Summary of Avian Influenza in Humans

In the past several years, it has become clear that avian influenza viruses can infect humans.

Human Disease Caused by Avian Influenza Viruses

Situations in which avian influenza virus subtypes have been recognized to be transmitted to humans and cause disease are identified in the following table.

Human Cases of Avian Influenza
Year	Subtype	No. of Cases	Location	Comments
1996	H7N7	1	United Kingdom	The case-patient developed conjunctivitis after cleaning a duck house (see References: CDC: Avian influenza A virus infections of humans).
1997	H5N1	18 (6 deaths)	Hong Kong	Case-patients were linked to an outbreak of H5N1 in poultry. Sustained person-to-person transmission did not occur, and the outbreak stopped when all birds in the Hong Kong commercial poultry industry (about 1.4 million) were slaughtered (see References: Yuen 1998).
1999	H9N2	2 (children ages 4 yr, 13 mo)	Hong Kong	Both case-patients had been hospitalized with influenza-like illness and both recovered uneventfully (see References: Peiris 1999, Uyeki 2002). No additional cases of person-to-person transmission occurred. Further investigation demonstrated that H9N2 strains were circulating in poultry in Hong Kong and China, although the viruses were not highly pathogenic for birds.
2002	H7N2	1	United States (Virginia)	Evidence of infection was found in one person in Virginia following a poultry outbreak (see References: CDC: Avian influenza A virus infections of humans).
2003	H5N1	2 (1 death)	Hong Kong	The 2 case-patients were family members who had recently traveled to China (see References: CDC: Avian influenza infection in humans). A third family member died while in China of an undiagnosed respiratory illness. No direct link between these cases and H5N1infection in poultry was identified.
2003	H7N7	89 (1 death)	The Netherlands	During an outbreak of H7N7 avian influenza in poultry, infection spread to poultry workers and their families in the area (see References: Fouchier 2004, Koopmans 2004, Stegeman 2004). Most patients had conjunctivitis, and several complained of influenza-like illness. The death occurred in a 57-year-old veterinarian. Subsequent serologic testing demonstrated that additional case-patients had asymptomatic infection.
2003	H7N2	1	New York	The source of exposure was not determined (see References: CDC 2004: Influenza activity).
2003	H9N2	1 (child)	Hong Kong	The source of infection remains unknown (see References: CDC 2004: Influenza activity).
2003-2008 (ongoing)	H5N1	More than 380, with a case-fatality rate >60%, according to official WHO numbers	Azerbaijan, Bangladesh, Cambodia, China, Djibouti, Egypt, Indonesia, Iraq, Lao PDR, Myanmar, Nigeria, Pakistan, Thailand, Turkey, Vietnam	Human cases are associated with an ongoing extensive outbreak of avian influenza in poultry (see References: WHO: Cumulative number of confirmed human cases of avian influenza A (H5N1). More information on this situation can be found in the section below.
2004	H7N3	2	Canada (British Columbia)	Two poultry workers became ill during an outbreak of H7N3 avian influenza in poultry (see References: Health Canada 2004). Both had conjunctivitis.
2004	H10N7	2 (infants)	Egypt	One child's father was a poultry merchant (see References: NIAID: Timeline of human flu pandemics).
2006	H7N3	1	United Kingdom	A poultry worker became ill during an outbreak that involved several farms (see May 1, 2006, CIDRAP News story).
2007	H7N2	4	United Kingdom	Case-patients were associated with a poultry outbreak of H7N2 in Wales (see References: CDC: Avian influenza A virus infections of humans). The case-patients had conjunctivitis and influenza-like illness.
2007	H9N2	1 (infant)	Hong Kong	The source of infection is unknown, although the child had visited a bird market with her parents before illness onset (see Mar 28, 2007, CIDRAP News story).

Information on Human Exposure to Avian Influenza Viruses

A serologic survey of 39 duck hunters and 68 wildlife professionals in Iowa conducted in late 2004 and early 2005 found that one duck hunter and two wildlife workers had serologic evidence of past infection to avian influenza virus H11N9. All three had extensive exposure to wild ducks and geese (see References: Gill 2006).

Another study of 42 veterinarians showed that the veterinarians were significantly more likely to have antibodies to avian influenza subtypes H5, H6, and H7 (indicating past infection with these viruses) compared with a group of 66 healthy nonveterinarian control subjects (see References: Myers 2007). Furthermore, veterinarians who had examined birds had a higher likelihood of having increased antibodies to the three avian subtypes compared with veterinarians who did not have exposure to birds.

A recent study from California involved sampling wild birds and marine mammals for avian influenza viruses from October 2005 through August 2007. The authors then estimated human-wildlife contact based on the prevalence of infection in the bird populations (see References: Siembieda 2008). The investigators defined three levels of contact (casual, recreational, and occupational) and sampled corresponding bird populations. The bird populations included the following: for casual contact, periurban species (eg, sparrows, crows, finches); for recreational contact, hunter-killed waterfowl; and for occupational contact, wild birds and sea mammals admitted to three wildlife hospitals in northern California. The authors found that waterfowl hunters (ie, those with recreational exposure) were eight times more likely to have contact with infected wildlife than those with casual or occupational exposures.

The Current Outbreak of H5N1 in Birds and Other Animals

An outbreak of HPAI caused by a strain of H5N1 avian influenza started in Asia in the fall of 2003 and spread in domestic poultry farms at an historically unprecedented rate. The outbreak tapered off in spring 2004 but in summer re-emerged in several countries in Asia (including Cambodia, China, Lao PDR, Thailand, and Vietnam), where it is ongoing.

In the summer of 2005, H5N1 began expanding its geographic range beyond Asia; this trend has continued into 2008 (see References: WHO 2008: H5N1 avian influenza: timeline of major events). For detailed information on avian influenza, see the document, Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations, on this Web site.

Areas affected by H5N1 avian influenza in poultry or migratory birds as of May 2008 are shown in the following table (see References: FAO 2008).

Countries Affected by H5N1 in Poultry and Wild Birds as of May 2008

East Asia,
Southeast Asia

Europe

Siberia, Central Asia, Middle East

Africa

Cambodia
China
Hong Kong
Indonesia
Japan
Lao PDR
Malaysia
Myanmar
Mongolia
South Korea
Thailand
Vietnam

Albania
Austria
Bosnia-Herzegovina
Bulgaria
Croatia
Czech Republic
Denmark
France
Germany
Greece
Hungary
Italy
Poland
Romania
Russia (European
Russia)
Scotland
Serbia
Slovakia
Slovenia
Spain
Sweden
Switzerland

United Kingdom

Afghanistan
Azerbaijan
Bangladesh
Cyprus
Georgia (former Soviet
republic)
India
Iran
Iraq
Israel
Jordan
Kazakhstan
Kuwait
Pakistan
Turkey
Ukraine
Russia (Siberia)
Saudi Arabia

West Bank and Gaza Strip

Benin
Burkina Faso
Cameroon
Djibouti
Egypt
Ghana
Ivory Coast
Niger
Nigeria
Sudan
Togo

H5N1 in Humans: Epidemiologic Features

Case Occurrence

The WHO has officially recognized more than 380 human cases of H5N1 influenza; cases have been reported from Azerbaijan, Bandladesh, Cambodia, China, Djibouti, Egypt, Indonesia, Iraq, Myanmar, Lao PDR, Nigeria, Pakistan, Thailand, Turkey, and Vietnam (see References: WHO: Cumulative number of confirmed human cases of avian influenza A [H5N1]; WHO: Situation updates).

An epidemiologic report on 340 confirmed H5N1 influenza cases published by the WHO in January 2008 demonstrated that the median age of cases was 18 years and that 90% of infections occurred in persons under 40 years of age (see References: WHO: Writing Committee of the Second World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A [H5N1] Virus 2008).

The overall case-fatality rate was 61% and was highest among persons 10 to 19 years of age and lowest among persons 50 years of age or older.
Of six infected pregnant women, four died and two had a spontaneous abortion.
Clusters of illness with at least two epidemiologically linked cases have been identified in 10 countries and have accounted for about 25% of all cases. Most clusters have involved two or three people and most have occurred among blood relatives; the largest cluster involved eight people (though the index case did not involve confirmatory tests). Persons involved in case clusters probably acquired infection from common-source exposures to poultry, but person-to-person transmission has occurred on occasion.

Exposure Information

Most cases have involved direct contact with poultry. Types of exposures that have been identified to date include:

Slaughtering, plucking, and preparing diseased birds
Handling fighting cocks or ducks that appear to be well
Playing with or holding diseased or dead poultry
Consumption of raw or undercooked poultry or poultry products (such as duck blood)

Low perceived risk and high population exposures to live chickens appear to be factors that are contributing to the spread of H5N1 from infected birds to humans (see References: Fielding 2005). For example, a survey of households in an area of rural Thailand affected by avian influenza found that 74% of households surveyed owned live poultry (see References: Olsen 2005: Poultry-handling practices during avian influenza outbreak, Thailand).

A case-control study from Vietnam found that the following risk factors were independently associated with H5N1 infection (see References: Dinh 2006):

Preparing sick or dead poultry for consumption in the 7 days before illness onset
Having sick or dead poultry in the household in the 7 days before illness onset
Lack of an indoor water source

Following recognition of a case of avian influenza in a rural village in southern Cambodia in 2005, investigators conducted a retrospective survey of poultry deaths and a seroepidemiologic survey of villagers (see References: Vong 2006). Of 194 households in the area, interviews were completed for 163; 155 of these households raised chickens or ducks, and 42 households were likely to have had an outbreak of avian influenza between January and March 2005 in their poultry (based on high rates of illness and mortality among chickens). Serologic testing of villagers approximately 2 months after outbreaks in poultry did not demonstrate any recent H5N1 infections, despite close contact with birds likely to have been infected with H5N1. These findings illustrate the following: (1) H5N1 was not easily transmitted from birds to humans and (2) asymptomatic or mildly symptomatic human infections did not occur.

A cross-sectional serologic survey of 322 poultry workers in areas of Thailand where outbreaks of avian influenza had occurred during the previous 6 months did not detect any workers who met the WHO criteria for confirmed infection (see References: Hinjoy 2007). These findings support the perspective that H5N1 avian influenza virus is not easily transmitted from birds to humans. A similar study conducted in Nigeria also found no serologic evidence of H5N1 infection among poultry workers (see References: Ortiz 2007).

The first report of H5N1 disease in humans contracted through exposure to wild birds occurred in the spring of 2006 (see References: Gilsdorf 2006). The discovery was made in a cluster of human cases in Azerbaijan; family members denied any contact with ill domestic poultry, but many wild swans had died in the area and were thought to have played a role. In August 2006, the CDC released a set of guidelines for conducting surveillance on dead birds (see References: CDC: Interim guidance for states conducting avian mortality surveillance for West Nile virus (WNV) and/or highly pathogenic H5N1 avian influenza virus).

In approximately 25% of cases, the source of exposure remains unclear and environment-to-human transmission is considered a possibility (such as through contact with virus-contaminated fomites).

A recent case report suggests that food markets with live birds may be a source of exposure for avian influenza (see References: Wang 2006).
Another report from China involving six cases with no obvious exposure to sick poultry found that all six had visited live poultry markets before illness onset (see References: Yu 2007).

Person-to-Person Transmission

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: Family clustering of avian influenza A [H5N1]).

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, CIDRAP News story). An Indonesian 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.

Intensified surveillance in northern Vietnam suggests that the local strains are adapting to humans. These efforts have identified less severe cases, more infections in older adults, and a few family clusters that suggest person-to-person spread (see References: WHO Writing Committee of the WHO Consultation on Human Influenza A/H5 2005).

H5N1 in Humans: Clinical Features

Clinical Features

H5N1 influenza generally presents as a severe pneumonia that often progresses to acute respiratory distress syndrome (ARDS). The summary table below outlines clinical and laboratory features for H5N1 influenza cases reported to the WHO through mid-December 2007 (see References: WHO: Writing Committee of the Second World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A [H5N1] Virus 2008).

Clinical and Common Laboratory Features of Influenza A (H5N1) Disease at Hospital Admission

Vietnam, Thailand, and Cambodia

2004-05

(Clade 1)

Indonesia

2005-06

(Clade 2.1)

China

2005-06

(Clade 2.3)

Egypt

2006-07

(Clade 2.2)

Turkey, Azerbaijan

2006

(Clade 2.2)


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