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1. Influenza

2. Avian Influenza

3. Virology

4. Pathogenesis and Immunology

5. Pandemic Preparedness

6. Vaccines

7. Laboratory Findings

8. Clinical Presentation

9. Treatment and Prophylaxis

10. Drugs

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Amedeo Influenza


  

 

Laboratory Findings

Gert van Zyl

 

Introduction

Various diagnostic modalities have been developed since influenza virus was first characterised in 1933 (Webster 1998). These diagnostic techniques can be employed to confirm a clinical diagnosis. In this chapter the role of the most important of these tests will be discussed as well as their advantages and limitations. However the best diagnostic test has little value without appropriate good quality specimen collection and correct patient information.


Laboratory Diagnosis of Human Influenza

Appropriate Specimen Collection

Respiratory specimens

The timing of specimen collection is very important since the yield is the highest for respiratory specimens obtained within four days of onset of symptoms. Different types of respiratory specimens can be used. Nasal washes and nasopharyngeal aspirates tend to be more sensitive than pharyngeal swabs. In patients that are intubated, tracheal aspirates and bronchial lavages can be collected (WHO 2005a). Washes and aspirates should contain sufficient respiratory epithelium for immunofluorescence tests. Specimens without sufficient cells are however still suitable for other methods such as rapid antigen detection, virus isolation and reverse transcription-polymerase chain reaction (RT-PCR).

Swabs should be transported in virus transport medium to prevent desiccation.

All specimens should arrive at the laboratory as soon as possible to avoid any degradation. Transportation in virus transport medium on ice or with refrigeration at 2-8 degrees Celsius is recommended if any delay in transportation is expected.

Blood specimens

Blood (whole blood, serum) specimens are collected for the purpose of antibody serology (determining the presence of antibodies to influenza). Acute and convalescent serum samples 14 - 21 days apart should be collected to demonstrate a significant (at least fourfold) rise in strain-specific antibody titre.

 

Clinical Role and Value of Laboratory Diagnosis

Patient management

Rapid diagnosis is important if early therapeutic interventions with costly antiviral drugs are being considered - to be effective, these drugs need to be started within 48 hours after the onset of symptoms (WHO 2005a). Candidates for early treatment are patients with underlying conditions and an increased risk of serious complications (see chapter "Clinical Presentation"). In particular, the diagnosis of influenza in elderly patients makes the clinician aware of a substantial risk of secondary bacterial infections with Staphylococcus aureus, Haemophilus influenzae and Streptococcus pneumoniae.

In addition, rapid testing for influenza virus plays a role in hospital infection control in reducing the spread of infection from patient to patient or from infected health care workers to high risk patients. These tests can also be used to diagnose influenza in travellers or outbreaks in semi-closed communities such as cruise ships (WHO 2005a).

Finally, diagnosis of influenza has prognostic value in healthy young adults where the disease has a short and benign course.

Surveillance

Influenza sentinel surveillance employs a variety of test and there seems a lack of standardisation even within the European region (Meerhoff 2004). Different techniques have different advantages and disadvantages. Therefore combinations of tests are used for surveillance. Rapid direct techniques such as RT-PCR (Bigl 2002) or EIA enable the fast detection of epidemics and can be used to distinguish between influenza A or B. Isolation of the virus in embryonated chicken eggs or on cell culture is necessary to subtype the viruses. Haemagglutinin and neuraminidase subtypes are respectively determined by haemagglutination inhibition assay and RT-PCR. Sequencing of PCR products is used to establish the molecular epidemiology of circulating viruses. This together with interstrain haemagglutination inhibition titres enable WHO to recommend appropriate vaccines that will most likely be protective against the circulating influenza strains. Surveillance is also important for public health policies since the health impact of a particular epidemic and cost benefit ratios of interventions such as vaccination can motivate policy makers to prioritise influenza prevention.


Laboratory Tests

Many factors should be considered in deciding which tests to use. Sensitivity, specificity, turn-around-time, repeatability, ease of performance and costs should all be taken into account. RT-PCR is generally more sensitive than serology and culture and the combination of RT-PCR with serology more sensitive than the combination of any other two methods (Zambon 2001). The sensitivity of culture is largely dependent on the laboratory where it is performed. Serology tends to be less expensive than RT-PCR but as it necessitates acute and convalescent blood specimens, diagnosis is only retrospective. Traditional culture is time-consuming but shell vial culture techniques allow diagnosis within 48-72 hours.

Direct Methods

Different methods exist for direct detection of influenza viruses. Some methods such as enzyme immunoassays (EIAs) can be suitable for bedside testing, others such as direct immunofluorescence allow for the preparation of slides onsite in clinics and posting of fixed slides to a central laboratory (Allwinn 2002). RT-PCR can only be performed in well equipped laboratory facilities by trained personnel. These methods can either detect both influenza A and B or differentiate between types (influenza A or B). The only direct technique that has the potential to differentiate between subtypes (i.e. on the basis of haemagglutinin and neuraminidase) is RT-PCR.

Immunofluorescence

For direct immunofluorescence, potentially infected respiratory epithelial cells are fixed to a slide and viral antigens contained in the cells is detected by specific antibodies which are either directly conjugated to a fluorescent dye (direct immunofluorescence) or detected by anti-antibodies linked to a fluorescent dye (indirect immunofluorescence). In both cases reactions are visualised under the fluorescence microscope and positive cells are distinguished on colour intensity and morphology of fluorescent areas. Direct immunofluorescence tends to allow faster results but is generally less sensitive than indirect immunofluorescence. Indirect immunofluorescence also has the advantage that pooled antisera can be used to screen for viral infection using a single anti-antibody conjugated to a fluorescent dye (fluorescein isothiocyanate-conjugated anti-mouse antibodies are commonly used; Stevens 1969). Immunofluorescence allows for the rapid diagnosis of respiratory specimens as long as sufficient respiratory epithelial cells are present in the specimens. However, inter-individual variation in reporting of immunofluorescence tests exists since interpretation is subjective and accuracy depends on the competence and experience of the operator.

Enzyme immuno assays or Immunochromatography assays

Enzyme immunoassays (EIAs) utilise antibodies directed against viral antigen that are conjugated to an enzyme. An incubation step with a chromogenic subtrate follows and a colour change is indicative of the presence of viral antigen. Certain enzyme immunoassays as well as similar assays using immunochromatography allow for bedside testing (Allwinn 2002) taking 10-30 minutes. These rapid assays are generally more expensive than direct immunofluorescence or virus culture. Sensitivities of EIAs vary between 64% and 78% (Allwinn 2002). Different rapid tests can detect either influenza A or B virus without distinguishing the type, influenza A virus only or detect both influenza A and B and identify the type. However non of these rapid tests can differentiate between subtypes that infect humans (H1N1 and H3N2) or avian influenza subtypes (FDA, 2005). A list of rapid tests that are available can be obtained from the following link: http://www.cdc.gov/flu/professionals/labdiagnosis.htm.

Reverse transcription polymerase chain reaction (RT-PCR)

RT-PCR is a process whereby RNA is first converted to complementary DNA (cDNA) and a section of the genome is then amplified through the use of primers that bind specifically to this target area. This allows for exponential amplification of small amounts of nucleic acid, through the action of a thermo stable DNA polymerase enzyme, which enables highly sensitive detection of minute amounts of viral genome.

Not only does RT-PCR have superior sensitivity (Steininger 2002) but it can also be used to differentiate between subtypes and conduct phylogenetic analysis (Allwinn 2002). RNA degradation of archival samples can decrease the sensitivity of RT-PCR (Frisbie 2004). Therefore specimens should be processed as fast as possible after collection.


Isolation Methods

Virus isolation or culture is a technique whereby a specimen is inoculated in a live culture system and the presence of virus infection is then detected in this culture system. Since culture amplifies the amount of virus it is more sensitive than direct methods with the exception of RT-PCR (that also employs amplification). Virus isolation is only of use if the live system or cells are sensitive for the virus that one intends to isolate.

Isolation requires the rapid transport of specimens to the laboratory since delays may lead to inactivation of virus (Allwinn 2002).

Embryonated egg culture

Specimens are inoculated into the amniotic cavity of 10-12 day embryonated chicken eggs. High yields of virus can be harvested after 3 days of incubation (WHO 2005d).

Since this technique requires the supply of fertilized chicken eggs and special incubators it is no longer used for the routine diagnosis of influenza infection. However egg isolation provides high quantities of virus and is a very sensitive culture system. Reference laboratories therefore utilise this culture system to ensure high sensitivity and to enable the production of virus stocks for epidemiological monitoring.


Cell Culture

Conventional culture: Various cell-lines are utilised to isolate influenza viruses, most commonly primary monkey kidney cells and Madin-Darby canine kidney (MDCK) cells. Some authors recommend the use of trypsin to aid virus entry into the cell lines (WHO 2005d). Conventional cell culture takes up to two weeks but has a very high sensitivity. Cytopathic effects such as syncytia and intracytoplasmic basophilic inclusion bodies are observed. The presence of influenza virus can be ascertained using haemadsorption using guinea pig red blood cells (Weinberg 2005) or immunofluorescence on cultured cells. The latter can also be used to type the isolated virus. Immunofluorescence has a higher sensitivity in detection of positive cultures than haemadsorption.

Shell vial culture: Shell vial culture allows for diagnosis within 48 hours (Allwinn 2002). This is brought about by centrifugation of the inoculum onto the cell culture monolayer and the performance of immunofluoresence before a cytopathic effect can be observed. Shell vial culture can however be less sensitive than conventional culture (Weinberg 2005).

Laboratory animals

Ferrets are often used in research facilities as a model of human influenza infection but have no role in routine diagnosis.


Serology

Serology refers to the detection of influenza virus-specific antibodies in serum (or other body fluids).

Serology can either detect total antibodies or be class-specific (IgG, IgA, or IgM).

Different serological techniques are available for influenza diagnosis: haemagglutination inhibition (HI), compliment fixation (CF), enzyme immunoassays (EIA) and indirect immunofluorescence.

Serological diagnosis has little value in diagnosing acute influenza. In order to diagnose acute infection, an at least four-fold rise in titre needs to be demonstrate, which necessitates both an acute and a convalescent specimen. However it may have value in diagnosing recently infected patients.

Serology is also used to determine the response to influenza vaccination (Prince 2003).

Serology has greater clinical value in paediatric patients without previous exposure to influenza since previous exposure can lead to heterologous antibody responses (Steininger 2002).

Haemagglutination inhibition (HI)

HI assays are labour intensive and time consuming assays that require several controls for standardisation. However the assay reagents are cheap and widely available. Various red blood cells such as guinea pig, fowl and human blood group "O" erythrocytes are used. An 0.4- 0.5% red blood cell dilution is generally used. Serum is pre-treated to remove non-specific haemagglutinins and inhibitors. A viral haemagglutinin preparation that produces visible haemagglutination (usually 4 haemagglutination units) is then pre-incubated with two-fold dilutions of the serum specimen. The lowest dilution of serum that inhibits haemagglutination is the HI titre. HI is more sensitive than complement fixation (Julkunen 1985, Prince 2003) and has the added advantage that it is more specific in differentiating between HA subtypes (Julkunen 1985).

Complement fixation (CF)

Complement fixation tests are based on the ability of antigen-antibody complexes to consume complement - which results in no compliment being available to lyse sensitised sheep red blood cells. These assays are labour intensive and necessitate controls for each procedure but reagents are cheap and widely available. CF assays are less sensitive than HI both in the diagnosis of acute infection and the determination of immunity after vaccination (Prince 2003)

Ezyme immuno assays (EIA)

EIAs are more sensitive than HI or CF assays (Bishai 1978, Julkunen 1985). Various commercial EIAs are available. Assays that detect IgG and IgA are more sensitive than IgM assays (Julkunen 1985) but are not indicative of acute infection.

Indirect immunofluorescence

Indirect immunofluorescence is not commonly used as a method to detect influenza virus antibodies.


Rapid Tests

The clinical value of a diagnostic test for influenza is to a large extent dependent on the particular test's turnaround time. The first diagnostic tests that were developed for influenza diagnosis were virus isolation and serological assays. At that stage it took more than two weeks to exclude influenza infection. Although shell vial tests have reduced the turn-around time of isolation, they are not generally regarded as rapid tests.

The development of direct tests such as immunofluorescence enabled the diagnosis within a few hours (1 to 2 incubation and wash steps). Immunofluorescence tests however necessitate skilled laboratory workers and the availability of immunofluorescence microscopes.

The revolution in rapid diagnosis of influenza was brought about by the development of rapid antigen assays (most of which work on an EIA or immunochromatography principle). These assays enable the diagnosis of influenza within 10-30 minutes. Some of these tests are so easy to perform that even non-laboratory trained people can perform these tests in the clinic, which is referred to as bedside or point-of-care testing.

RT-PCR reactions that required a gel electrophoresis step were initially time consuming but the relatively recent development of real-time technology made RT-PCR diagnosis within about two hours possible. Although antigen assays are generally the most user-friendly, they are not as sensitive as direct immunofluorescence, isolation or RT-PCR.

Table 1 compares the characteristics of the different test methods available for influenza diagnosis.


Table 1: Comparison of test characteristics*

Test

Sensitivity

Turnaround time

Ease of performance

Affordability

Direct detection

       

Rapid tests (EIA / chromatography)

-2

+2

+2

0

Immunofluorescence

0

+1

+1

+1

Gel electrophoresis RT-PCR

+2

0

-1

-2

Real-time RT-PCR

+2

+1

-1

-2

Viral culture

       

Routine viral culture

+2

-2

-1

+2

Shell vial culture

+1

0

-1

+1

Serology

       

EIA

+2

-2

+1

+1

Haemagglutination inhibition

+1

-2

-1

+2

Complement fixation

0

-2

-2

+2


*Relative criteria for favourability of tests (5 point ordinal scale)
-2: very unfavourable characteristic
-1: unfavourable characteristic
0: average characteristic
+1: favourable characteristic
+2: very favourable characteristic


Differential diagnosis of flu-like illness

Many different symptoms are described as influenza-like: fever, cough, nasal congestion, headache, malaise and myalgia. However no clear definition or uniformity in the use of the term "flu-like" exists.

During an epidemic the clinical symptoms of fever, cough, severe nasal symptoms and loss of appetite are highly predictive of influenza (Zambon 2001). However many other infections can present with influenza-like symptoms. These include viral, bacterial, mycoplasmal, chlamydial and fungal infections and also parasite infestations. Infections that could either be life-threatening also in the young and healthy, such as viral haemorrhagic fevers, or infections such as legionellosis that are life-threatening in at-risk groups such as the old-aged, can initially present with flu-like symptoms. Therefore it is important to consider a wide differential diagnosis which should be guided by the patient's history, which includes travel, occupational exposure, contact with animals and sick individuals, history of symptoms as well as the local epidemiology of disease.


Diagnosis of suspected human infection with an avian influenza virus

Introduction

Accurate and rapid clarification of suspected cases of H5N1 infection by laboratory diagnosis is of paramount importance in the initiation and continuation of appropriate treatment and infection control measures. Isolation of virus from specimens of suspected cases of avian influenza should be conducted in specialised reference laboratories with at least Biosafety Level 3 facilities.

Specimen Collection

Specimens for virus detection or isolation should be collected within 3 days after the onset of symptoms and rapidly transported to the laboratory. A nasopharyngeal aspirate, nasal swab, nasal wash, nasopharyngeal swab, or throat swab are all suitable for diagnosis. However a nasopharyngeal aspirate is the specimen of choice. In cases where patients are intubated, a transtracheal aspirates and a bronchoalveolar lavage can be collected.

At the same time, acute and convalescent serum samples should be collected for serological diagnosis (WHO 2005b).

Virological Diagnostic Modalities

Rapid identification of the infecting agent as an influenza A virus can be performed by ordinary influenza rapid tests that differentiate between types. However commercial rapid chromatographic methods have a sensitivity of only 70% for avian influenza compared to culture (Yuen 2005). Direct diagnosis of influenza H5N1 infection can be performed by indirect immunofluorescence on respiratory cells fixed to glass slides using a combination of influenza type A/H5-specific monoclonal antibody pool, influenza A type specific and influenza B type-specific monoclonal antibody pools as well as influenza A/H1 and an A/H3 specific monoclonal antibody (available from WHO) and anti-mouse FITC for the detection step. This assay allows for the rapid differentiation of human H5 influenza infection from other influenza types and subtypes but cannot exclude H5N1 infection due to lack of sensitivity. Therefore culture and/ or RT-PCR that are more sensitive should also be performed.

Virus can be isolated in embryonated chicken eggs, Madin Darby canine kidney (MDCK) cells or Rhesus monkey kidney cells (LLC-MK2) (de Jong 2005, Yuen 2005). Other common cell lines such as Hep-2 or RD cells are also permissible to avian influenza A/H5 virus. Cytopathic effects are non-specific and influenza A virus infection of cells can be detected by immunofluorescence for nucleoprotein. HI of cell culture supernatant, H5-specific immunofluorescence (using monoclonal antibodies against H5) or RT-PCR can be used to subtype these viruses. Primers are available to detect both H5 and N1 genes of avain influenza by RT-PCR (WHO 2005c). H9-specific primers are also available (WHO 2005c)

Detection of Influenza A/H5 by real-time RT-PCR offers a rapid and highly sensitive method to diagnose H5N1 infection (Ng 2005).

Serology: A fourfold rise in titre from acute to convalescent specimens is also diagnostic of infection in patients that recovered (Yuen 2005).

Other Laboratory Findings

Leucopenia and especially lymphopenia (which has been shown to be a sign of poor prognosis in patients from Thailand), thrombocytopenia and moderately elevated transaminase levels are common findings (Beigel 2005).


New developments and the future of influenza diagnostics

A few trends in influenza diagnosis have been observed. The availability of anti-influenza drugs which must be given early in infection in order to be effective has emphasised the need for early diagnosis which stimulated the development of many EIA or immunochromotographic rapid tests with such low complexity that they enable bedside testing. Yet these tests' value is limited by their relatively low sensitivity especially for the diagnosis of avian influenza.

Real-time RT-PCR offers a highly sensitive and specific alternative. Technological developments are making real-time RT-PCR more widely available since instruments are becoming smaller, more efficient and user-friendly. Therefore real-time RT-PCR has already gained prominence in influenza pandemic preparedness since it will enable laboratories to make a rapid sensitive and specific diagnosis of human cases of avian influenza. The only remaining hurdle remains its relative high cost; but the highly competitive market has already made these tests more affordable.


Conclusion

Molecular diagnostic techniques play a more and more prominent role in laboratory diagnosis of influenza. Direct rapid tests have also become an important tool for investigating influenza-like illness.

Viral culture however remains important especially for reference laboratories since it is cheap, sensitive and enables characterisation of viruses. Furthermore unlike molecular testing it is "unbiased" and can detect the unexpected new strain.

Influenza serology's main value lies in epidemiological investigations of yearly epidemics, avian to human transmissions and drug and vaccine trials. It has limited value for routine diagnosis.

We can thus conclude that virological diagnosis for influenza has value for the individual patient, epidemiological investigations and infection control. The appropriate selection of a particular test will is determinded by the test characteristics and the specific diagnostic or public health needs.

A positive diagnostic test is the difference between someone with flu-like illness and a definite diagnosis of influenza or between a suspected human case of avian influenza and a confirmed case.


 
 
 



Useful Internet sources relating to Influenza Diagnosis

http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5408a1.htm

http://www.fda.gov/cdrh/oivd/tips/rapidflu.html

http://www.who.int/csr/disease/avian_influenza/guidelines/RapidTestInfluenza_web.pdf

http://www.who.int/csr/disease/avian_influenza/guidelines/humanspecimens/en/print.html

http://www.who.int/csr/disease/avian_influenza/guidelines/avian_labtests2.pdf

http://www.who.int/csr/resources/publications/influenza/whocdscsrncs20025rev.pdf


References

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