Treyfish
Moderator
A very good page on Avian influenza background,studies and implications:yinyang: Date: Wed 16 May 2007
Source: CIDRAP, Wed 16 May 2007 [edited]
Avian influenza (bird flu): implications for human disease
[A comprehensive reference document covering all aspects of the biology of
human avian influenza H5N1 virus updated to 16 May 2007 is available at the
CIDRAP (Center for Infectious Disease Research and Policy, University of
Minnesota) website via the general link:
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Agent>]
The contents are arranged under the following headings and include
references to key publications:
Agent
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Agent>
Laboratory testing for avian influenza in humans
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Laboratory_Testing_for_Influenza_in>
Summary of avian influenza in humans
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Summary_of_Avian_Influenza_in_Human>
Current outbreak of H5N1 in birds and other animals
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_The_Current_Outbreak_of_H5N1_in_Bir>
H5N1 in humans: epidemiologic features
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_H5N1_in_Humans:_Epidemiologic_Consi>
H5N1 in humans: clinical features
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_H5N1_in_Humans:>
Treatment and prophylaxis
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Treatment_and_Prophylaxis>
Current status of H5N1 candidate vaccines
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Current_Status_of>
Current WHO and CDC travel recommendations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Current_WHO_and_CDC_Travel_Recommen>
Use of seasonal flu vaccine in humans at risk for H5N1 infection
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Use_of_Seasonal>
Surveillance considerations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Surveillance_Considerations>
Influenza pandemic considerations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Influenza_Pandemic_Considerations_2>
Infection control recommendations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Infection_Control>
Guidance to protect workers from avian influenza viruses
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Guidance_to_Protect>
Food safety issues
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Food_Safety_Issues_1>
References
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_References_1>
The following sections are reproduced in full to give a flavour of the
content of the document, which uniquely attempts to cover the whole field
of the interaction of avian H5N1 influenza viruses with the human population.
The 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 5 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. A total of 8 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 (8
structural proteins and 2 nonstructural proteins). These include the
following: 3 transcriptases (PB2, PB1, and PA), 2 surface glycoproteins
(hemagglutinin [HA] and neuraminidase [NA]), 2 matrix proteins (M1 and M2),
one nucleocapsid protein (NP), and 2 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 9 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 3 subtypes of HA (H1, H2,
and H3) and 2 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,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/aug3106dog.html>
CIDRAP News story).
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).
* 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 2 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 per cent. 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, Europe, and Africa.
* Recent genetic characterization of H5N1 strains involved in the current
panzootic has demonstrated 2 distinct phylogenetic clades (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 prepandemic vaccines). Clade
1 viruses have circulated primarily in Cambodia, Thailand, and Vietnam, and
clade 2 viruses have circulated primarily in China and Indonesia and have
spread westward to the Middle East, Europe, and Africa. A total of 6
different subclades of clade 2 have been recognized; 3 of these are
primarily responsible for recent human H5N1 cases. The most recent wave of
outbreaks in Thailand (in July 2006) has been caused by viruses closely
related to those that caused outbreaks in Thailand in 2004-2005 and to
viruses recently circulating in southeast China (see References:
Chutinimitkul 2007).
* H7 and H9 subtypes
* H7 includes HPAI and LPAI strains.
* H9 is only known to include LPAI strains.
* These subtypes have caused infections in humans on rare occasions (see
References: CDC: Avian influenza A viruses; NIAID: Timeline of human
pandemics).
* 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]).
* Physical characteristics of influenza A 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 of time 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 deg C [39.2
deg F] and was undetectable after 4 days at 22 deg C [71.6 deg F] (see
References: Webster 1978). Another study found that several avian influenza
viruses persisted in distilled water for 207 days at 17 deg C [62.6 deg F]
and 102 days at 28 deg C [82.4 deg F] (see References: Stallknecht 1990).
* Recent data from studies of H5N1 in domestic ducks have shown that H5N1
can survive in the environment for 6 days at 37 deg C [98.6 deg F] (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 deg C [132.8 deg F] for 3
hours or 60 deg C [140 deg F] or more for 30 minutes; acidic pH conditions;
presence of oxidizing agents such as sodium dodecyl sulfate, lipid
solvents, and B-propiolactone; and exposure to disinfectants such as
formalin and iodine compounds....AND "bleach"
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 a designated influenza reference laboratory for
confirmation and N-typing (see References: WHO: Guidelines for global
surveillance of influenza A/H5; WHO: Recommended laboratory tests to
identify avian influenza A virus in specimens from patients with an
influenza-like illness).
* 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 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 avian influenza and other
emerging influenza variants (see References: Weinberg 2005).
* Laboratory tests are required for specific identification of avian
influenza. The most likely ways that an avian influenza strain would be
detected in the human population are:
* Outbreak investigations or investigation of unexplained death in a
previously healthy individual
* Influenza surveillance with laboratory testing
* Investigation of unusual laboratory findings
* Testing of persons with influenza-like symptoms who meet certain exposure
criteria
* 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). 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:
* 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 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 2nd or 3rd 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 (4 deg C [39.2 deg F] to 8 deg C
[46.4 deg F]) until testing is performed. Freezing at 70 deg C [158 deg F]
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. A
total of 8 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
-------------------------
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 2005: 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 (e.g., probable
H1 or H3 isolates) to prevent recombination.
* Indirect immunofluroescence (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 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 USDA classifies 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 an Final Rule in the 18 Mar 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).
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
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 and mumps
viruses, may also cause haemadsorption. 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 (e.g., 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 percent of positive
specimens) to 7 days (100 percent 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.
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.
* RT-PCR assays
* RT-PCR assays use conserved targets such as the matrix (M) protein for
genus-level identification. Hemagglutinin and neuraminidase 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
percent to 100 percent 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 3 Feb 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.
* Multiplex real-time RT-PCR assays have been developed for specific
detection of H5N1 (See References: Kessler 2004, Ng 2005, Payungporn 2005).
* 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 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 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).
* There were 2 reports released in August 2006 which 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 2 rounds of testing showed
that the FluChip allowed users to obtain correct information about both
type and subtype from 72 percent of 72 samples tested. Full information on
type, but only partial information on subtype, was obtained for an
additional 13 percent of the samples, while 10 percent 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 3 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 percent and clinical specificity of
100 percent (see 15 Nov 2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/panflu/news/nov1506mchip.html>
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 (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 percent to 80 percent.
* 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 (i.e., 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 percent) 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).
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), 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 4-fold
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 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 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).
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.
* 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
Summary of avian influenza in humans
------------------------------------
In the past several years, it has become clear that avian influenza viruses
can infect humans. Situations where avian influenza virus subtypes have
been recognized to be transmitted to humans and cause disease. This section
comprises a tabulation of avian influenza in humans during the period 1997
to 2004.
Current outbreak of H5N1 in birds and other animals
---------------------------------------------------
The 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 People's Democratic Republic [PDR], Thailand, and Vietnam),
where it is ongoing. The H5N1 strains currently causing outbreaks across
Asia and elsewhere are genetically distinct from the strain isolated from
humans in Hong Kong in 1997. Since January 2002, the predominant avian H5N1
strain in southern China has been genotype Z. Since its emergence, this
strain has replaced other genotypes and has become the predominant genotype
circulating in aquatic and terrestrial poultry in the region (see
References: Li 2004). Ongoing market surveillance in China demonstrates
that a single sublineage of genotype Z (Fujian [FJ]-like) has emerged in
poultry in China since late 2005 (see References: Smith 2006). A 2006 study
suggests that the H5N1 virus has been circulating in southern China for
nearly a decade (see References: Chen 2006).
In the summer of 2005, H5N1 began expanding its geographic range beyond
Asia; this trend has continued into 2007. This section comprises a
comprehensive listing of the outbreaks of H5N1 avian influenza virus
infection. As of August 2006, more than 220 million birds have been killed
by the virus or culled to prevent further spread (see References: FAO:
Caucasus, Balkins at high risk for deadly H5N1 virus), and the number keeps
growing. Areas currently affected by H5N1 avian influenza in poultry or
migratory birds are shown in the following table.
Countries Affected by H5N1 in Poultry and Wild Birds as of 3 May 2007: East
Asia, Southeast Asia Europe Siberia, Central Asia, Middle East Africa
Cambodia China Hong Kong Indonesia Japan Lao DPR Malaysia Myanmar Mongolia
South Korea Thailand Vietnam Albania Austria Bosnia-Herzegovina Bulgaria
Croatia Czech Republic Denmark England France Germany Greece Hungary Italy
Poland Romania Russia (European Russia) Scotland Serbia Slovakia Slovenia
Spain Sweden Switzerland Afghanistan Azerbaijan Bangladesh Cyprus Georgia
(former Soviet republic) India Iran Iraq Israel Jordan Kazakhstan Kuwait
Pakistan Palestine Turkey Ukraine Russia (Siberia) Saudi Arabia Burkina
Faso Cameroon Djibouti Egypt Ghana Ivory Coast Niger Nigeria Sudan.
Information taken from FAO (see References: FAO 2006: Should wild birds now
be considered a permanent reservoir of the virus?) and current news reports
If H5N1 continues 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 in 2001 and 2003 (see References:
Cyranoski 2004), and human H3N2 influenza viruses are endemic in pigs in
that area. H5N1 has been reported in pigs in Indonesia as well (see Oct 10,
2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/oct1006pigs.html>
CIDRAP News Story and 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 may also increase. It is also possible
that a pandemic strain could emerge following a more gradual process of
adaptive mutation in humans, which is likely what happened with the 1918
H1N1 pandemic strain (see References: Taubenberger 2005; WHO: Influenza
pandemic preparedness and response 2005).
Domestic cats recently have been shown to be susceptible to H5N1 infection
and 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). Asymptomatic infection has been reported in
domestic cats (see References: Leschnik 2007). FAO recommends that avian
influenza in cats should be closely monitored (see References: FAO: 2007).
H5N1 in humans: epidemiologic features
--------------------------------------
WHO has officially recognized more than 300 human cases of H5N1 influenza;
cases have been reported from Azerbaijan, Cambodia, China, Djibouti, Egypt,
Indonesia, Iraq, Lao People's Democratic Republic, Nigeria, Thailand,
Turkey, and Vietnam (see References: WHO: Cumulative number of confirmed
human cases of avian influenza A [H5N1]; WHO: Situation updates).
The case fatality rate is higher than 50 per cent overall and higher than
70 per cent presently in Indonesia, according to WHO numbers. An
epidemiologic report on 256 confirmed H5N1 influenza cases published by WHO
in February 2007 demonstrated that the median age of cases was 18 years and
that 89 per cent of infections occurred in persons under 40 years of age
(see References: WHO: Update: Epidemiology of WHO-confirmed human cases of
avian A[H5N1] infection, 25 Nov 2003 - 24 Nov 2006). The overall case
fatality rate for this period was 60 per cent and the median number of days
from symptom onset to death was 9 days. Most recognized human cases have
involved direct contact with poultry (see References: WHO Writing Committee
of the WHO Consultation on Human Influenza A/H5 2005).
Types of exposures that have been identified to date include:
* Slaughtering, plucking and preparing diseased birds
* Handling fighting cocks
* Playing with poultry (particularly asymptomatic ducks)
* Consumption of duck blood and possibly undercooked poultry
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 per cent of households surveyed owned live poultry (see References:
Olsen 2005: Poultry-handling practices during avian influenza outbreak,
Thailand).
A recently published report of 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.
Another recent case report suggests that food markets with live birds may
be a source of exposure for avian influenza (see References: Wang 2006).
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 survey of 257 poultry workers in Italy
regarding knowledge, attitudes, and practices around avian influenza found
that workers in that area had a relatively low perceived risk of avian
influenza and did not routinely practice biosecurity measures (such as
wearing protective equipment and handwashing) (see References: Abbate 2006).
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, 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). 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
7 cases of person-to-person transmission; one of the cases involved 2
generations of transmission (see References: WHO: Avian influenza:
Situation in Indonesia: Update 14 and see 24 May 2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/may2406cluster.html>
CIDRAP News story).
An Indonesian official recently put the number of clusters in that country
at 10, all involving cases in blood relatives (see 12 Jan 2007,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/jan1607human2.html>
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).
The entire text is supplemented by comprehensive referencing of original
sources.
--
communicated by
ProMED-mail rapporteur Mary Marshall
[This document provides an invaluable and authoritative resource for
life-science professionals, health care personnel, and others seeking
information on the interaction of the H5N1 avian influenza viruses with the
human population. ProMED-mail thanks Mary Marshall for drawing attention to
the existence of this unique resource. - Mod.CP]:tiphat:
Source: CIDRAP, Wed 16 May 2007 [edited]
Avian influenza (bird flu): implications for human disease
[A comprehensive reference document covering all aspects of the biology of
human avian influenza H5N1 virus updated to 16 May 2007 is available at the
CIDRAP (Center for Infectious Disease Research and Policy, University of
Minnesota) website via the general link:
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Agent>]
The contents are arranged under the following headings and include
references to key publications:
Agent
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Agent>
Laboratory testing for avian influenza in humans
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Laboratory_Testing_for_Influenza_in>
Summary of avian influenza in humans
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Summary_of_Avian_Influenza_in_Human>
Current outbreak of H5N1 in birds and other animals
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_The_Current_Outbreak_of_H5N1_in_Bir>
H5N1 in humans: epidemiologic features
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_H5N1_in_Humans:_Epidemiologic_Consi>
H5N1 in humans: clinical features
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_H5N1_in_Humans:>
Treatment and prophylaxis
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Treatment_and_Prophylaxis>
Current status of H5N1 candidate vaccines
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Current_Status_of>
Current WHO and CDC travel recommendations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Current_WHO_and_CDC_Travel_Recommen>
Use of seasonal flu vaccine in humans at risk for H5N1 infection
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Use_of_Seasonal>
Surveillance considerations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Surveillance_Considerations>
Influenza pandemic considerations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Influenza_Pandemic_Considerations_2>
Infection control recommendations
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Infection_Control>
Guidance to protect workers from avian influenza viruses
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Guidance_to_Protect>
Food safety issues
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_Food_Safety_Issues_1>
References
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts/avflu_human.html#_References_1>
The following sections are reproduced in full to give a flavour of the
content of the document, which uniquely attempts to cover the whole field
of the interaction of avian H5N1 influenza viruses with the human population.
The 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 5 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. A total of 8 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 (8
structural proteins and 2 nonstructural proteins). These include the
following: 3 transcriptases (PB2, PB1, and PA), 2 surface glycoproteins
(hemagglutinin [HA] and neuraminidase [NA]), 2 matrix proteins (M1 and M2),
one nucleocapsid protein (NP), and 2 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 9 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 3 subtypes of HA (H1, H2,
and H3) and 2 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,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/aug3106dog.html>
CIDRAP News story).
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).
* 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 2 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 per cent. 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, Europe, and Africa.
* Recent genetic characterization of H5N1 strains involved in the current
panzootic has demonstrated 2 distinct phylogenetic clades (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 prepandemic vaccines). Clade
1 viruses have circulated primarily in Cambodia, Thailand, and Vietnam, and
clade 2 viruses have circulated primarily in China and Indonesia and have
spread westward to the Middle East, Europe, and Africa. A total of 6
different subclades of clade 2 have been recognized; 3 of these are
primarily responsible for recent human H5N1 cases. The most recent wave of
outbreaks in Thailand (in July 2006) has been caused by viruses closely
related to those that caused outbreaks in Thailand in 2004-2005 and to
viruses recently circulating in southeast China (see References:
Chutinimitkul 2007).
* H7 and H9 subtypes
* H7 includes HPAI and LPAI strains.
* H9 is only known to include LPAI strains.
* These subtypes have caused infections in humans on rare occasions (see
References: CDC: Avian influenza A viruses; NIAID: Timeline of human
pandemics).
* 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]).
* Physical characteristics of influenza A 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 of time 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 deg C [39.2
deg F] and was undetectable after 4 days at 22 deg C [71.6 deg F] (see
References: Webster 1978). Another study found that several avian influenza
viruses persisted in distilled water for 207 days at 17 deg C [62.6 deg F]
and 102 days at 28 deg C [82.4 deg F] (see References: Stallknecht 1990).
* Recent data from studies of H5N1 in domestic ducks have shown that H5N1
can survive in the environment for 6 days at 37 deg C [98.6 deg F] (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 deg C [132.8 deg F] for 3
hours or 60 deg C [140 deg F] or more for 30 minutes; acidic pH conditions;
presence of oxidizing agents such as sodium dodecyl sulfate, lipid
solvents, and B-propiolactone; and exposure to disinfectants such as
formalin and iodine compounds....AND "bleach"
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 a designated influenza reference laboratory for
confirmation and N-typing (see References: WHO: Guidelines for global
surveillance of influenza A/H5; WHO: Recommended laboratory tests to
identify avian influenza A virus in specimens from patients with an
influenza-like illness).
* 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 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 avian influenza and other
emerging influenza variants (see References: Weinberg 2005).
* Laboratory tests are required for specific identification of avian
influenza. The most likely ways that an avian influenza strain would be
detected in the human population are:
* Outbreak investigations or investigation of unexplained death in a
previously healthy individual
* Influenza surveillance with laboratory testing
* Investigation of unusual laboratory findings
* Testing of persons with influenza-like symptoms who meet certain exposure
criteria
* 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). 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:
* 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 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 2nd or 3rd 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 (4 deg C [39.2 deg F] to 8 deg C
[46.4 deg F]) until testing is performed. Freezing at 70 deg C [158 deg F]
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. A
total of 8 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
-------------------------
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 2005: 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 (e.g., probable
H1 or H3 isolates) to prevent recombination.
* Indirect immunofluroescence (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 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 USDA classifies 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 an Final Rule in the 18 Mar 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).
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
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 and mumps
viruses, may also cause haemadsorption. 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 (e.g., 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 percent of positive
specimens) to 7 days (100 percent 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.
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.
* RT-PCR assays
* RT-PCR assays use conserved targets such as the matrix (M) protein for
genus-level identification. Hemagglutinin and neuraminidase 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
percent to 100 percent 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 3 Feb 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.
* Multiplex real-time RT-PCR assays have been developed for specific
detection of H5N1 (See References: Kessler 2004, Ng 2005, Payungporn 2005).
* 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 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 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).
* There were 2 reports released in August 2006 which 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 2 rounds of testing showed
that the FluChip allowed users to obtain correct information about both
type and subtype from 72 percent of 72 samples tested. Full information on
type, but only partial information on subtype, was obtained for an
additional 13 percent of the samples, while 10 percent 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 3 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 percent and clinical specificity of
100 percent (see 15 Nov 2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/panflu/news/nov1506mchip.html>
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 (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 percent to 80 percent.
* 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 (i.e., 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 percent) 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).
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), 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 4-fold
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 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 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).
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.
* 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
Summary of avian influenza in humans
------------------------------------
In the past several years, it has become clear that avian influenza viruses
can infect humans. Situations where avian influenza virus subtypes have
been recognized to be transmitted to humans and cause disease. This section
comprises a tabulation of avian influenza in humans during the period 1997
to 2004.
Current outbreak of H5N1 in birds and other animals
---------------------------------------------------
The 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 People's Democratic Republic [PDR], Thailand, and Vietnam),
where it is ongoing. The H5N1 strains currently causing outbreaks across
Asia and elsewhere are genetically distinct from the strain isolated from
humans in Hong Kong in 1997. Since January 2002, the predominant avian H5N1
strain in southern China has been genotype Z. Since its emergence, this
strain has replaced other genotypes and has become the predominant genotype
circulating in aquatic and terrestrial poultry in the region (see
References: Li 2004). Ongoing market surveillance in China demonstrates
that a single sublineage of genotype Z (Fujian [FJ]-like) has emerged in
poultry in China since late 2005 (see References: Smith 2006). A 2006 study
suggests that the H5N1 virus has been circulating in southern China for
nearly a decade (see References: Chen 2006).
In the summer of 2005, H5N1 began expanding its geographic range beyond
Asia; this trend has continued into 2007. This section comprises a
comprehensive listing of the outbreaks of H5N1 avian influenza virus
infection. As of August 2006, more than 220 million birds have been killed
by the virus or culled to prevent further spread (see References: FAO:
Caucasus, Balkins at high risk for deadly H5N1 virus), and the number keeps
growing. Areas currently affected by H5N1 avian influenza in poultry or
migratory birds are shown in the following table.
Countries Affected by H5N1 in Poultry and Wild Birds as of 3 May 2007: East
Asia, Southeast Asia Europe Siberia, Central Asia, Middle East Africa
Cambodia China Hong Kong Indonesia Japan Lao DPR Malaysia Myanmar Mongolia
South Korea Thailand Vietnam Albania Austria Bosnia-Herzegovina Bulgaria
Croatia Czech Republic Denmark England France Germany Greece Hungary Italy
Poland Romania Russia (European Russia) Scotland Serbia Slovakia Slovenia
Spain Sweden Switzerland Afghanistan Azerbaijan Bangladesh Cyprus Georgia
(former Soviet republic) India Iran Iraq Israel Jordan Kazakhstan Kuwait
Pakistan Palestine Turkey Ukraine Russia (Siberia) Saudi Arabia Burkina
Faso Cameroon Djibouti Egypt Ghana Ivory Coast Niger Nigeria Sudan.
Information taken from FAO (see References: FAO 2006: Should wild birds now
be considered a permanent reservoir of the virus?) and current news reports
If H5N1 continues 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 in 2001 and 2003 (see References:
Cyranoski 2004), and human H3N2 influenza viruses are endemic in pigs in
that area. H5N1 has been reported in pigs in Indonesia as well (see Oct 10,
2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/oct1006pigs.html>
CIDRAP News Story and 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 may also increase. It is also possible
that a pandemic strain could emerge following a more gradual process of
adaptive mutation in humans, which is likely what happened with the 1918
H1N1 pandemic strain (see References: Taubenberger 2005; WHO: Influenza
pandemic preparedness and response 2005).
Domestic cats recently have been shown to be susceptible to H5N1 infection
and 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). Asymptomatic infection has been reported in
domestic cats (see References: Leschnik 2007). FAO recommends that avian
influenza in cats should be closely monitored (see References: FAO: 2007).
H5N1 in humans: epidemiologic features
--------------------------------------
WHO has officially recognized more than 300 human cases of H5N1 influenza;
cases have been reported from Azerbaijan, Cambodia, China, Djibouti, Egypt,
Indonesia, Iraq, Lao People's Democratic Republic, Nigeria, Thailand,
Turkey, and Vietnam (see References: WHO: Cumulative number of confirmed
human cases of avian influenza A [H5N1]; WHO: Situation updates).
The case fatality rate is higher than 50 per cent overall and higher than
70 per cent presently in Indonesia, according to WHO numbers. An
epidemiologic report on 256 confirmed H5N1 influenza cases published by WHO
in February 2007 demonstrated that the median age of cases was 18 years and
that 89 per cent of infections occurred in persons under 40 years of age
(see References: WHO: Update: Epidemiology of WHO-confirmed human cases of
avian A[H5N1] infection, 25 Nov 2003 - 24 Nov 2006). The overall case
fatality rate for this period was 60 per cent and the median number of days
from symptom onset to death was 9 days. Most recognized human cases have
involved direct contact with poultry (see References: WHO Writing Committee
of the WHO Consultation on Human Influenza A/H5 2005).
Types of exposures that have been identified to date include:
* Slaughtering, plucking and preparing diseased birds
* Handling fighting cocks
* Playing with poultry (particularly asymptomatic ducks)
* Consumption of duck blood and possibly undercooked poultry
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 per cent of households surveyed owned live poultry (see References:
Olsen 2005: Poultry-handling practices during avian influenza outbreak,
Thailand).
A recently published report of 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.
Another recent case report suggests that food markets with live birds may
be a source of exposure for avian influenza (see References: Wang 2006).
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 survey of 257 poultry workers in Italy
regarding knowledge, attitudes, and practices around avian influenza found
that workers in that area had a relatively low perceived risk of avian
influenza and did not routinely practice biosecurity measures (such as
wearing protective equipment and handwashing) (see References: Abbate 2006).
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, 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). 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
7 cases of person-to-person transmission; one of the cases involved 2
generations of transmission (see References: WHO: Avian influenza:
Situation in Indonesia: Update 14 and see 24 May 2006,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/may2406cluster.html>
CIDRAP News story).
An Indonesian official recently put the number of clusters in that country
at 10, all involving cases in blood relatives (see 12 Jan 2007,
<http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/biofacts//cidrap/content/influenza/avianflu/news/jan1607human2.html>
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).
The entire text is supplemented by comprehensive referencing of original
sources.
--
communicated by
ProMED-mail rapporteur Mary Marshall
[This document provides an invaluable and authoritative resource for
life-science professionals, health care personnel, and others seeking
information on the interaction of the H5N1 avian influenza viruses with the
human population. ProMED-mail thanks Mary Marshall for drawing attention to
the existence of this unique resource. - Mod.CP]:tiphat: