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Diagnosing Human Cases of Avian Influenza

Bird flu viruses are commonly diagnosed by one of two methods – a PCR test or a viral antigen detection test.

PCR

A PCR test is the abbreviation of a polymerase chain reaction test where samples of DNA - blood, tissue or other bodily fluids are taken from the suspected victim for analysis. By adding a catalyst known as a polymerase enzyme, the DNA quickly replicates itself to form a reasonable sized sample that can then be examined for the presence of any single stranded RNA strands typical to bird flu viruses. This can specifically identify if avian influenza viruses are present, but not the strain or subtype of bird flu contracted.

Viral Antigen Detection

In this test, samples of tissue are taken by scraping small amounts of skin or swabbing samples of throat mucus from the patient. Infected cells present within the sample will possess antigens on its surface produced by infection from avian influenza viruses. To combat antigens, the human body naturally produces corresponding antibodies that target and combat the antigens of a virus.

By releasing labeled bird flu antibodies into the sample, the presence of any antigens (and subsequently a virus) can be verified through observing whether the antibodies react with the sample. If the antibodies attach to the cell surfaces, then this indicates a positive result and that the sample is in fact infected.

Virus Culture

To determine the specific details of the virus strain and pathogenicity, scientists must further cultivate the virus sample in living cells (commonly canine kidney cells) to allow for growth, microscope examination and observation of the virus' clinical effects. This process, a form of cell culture, yields a more thorough result on the virus, including pathogenicity and mortality rate. The WHO rates virus culture as the gold testing standard for avian influenza.

Time Taken for Diagnosis

PCR tests typically require 3 hours in the laboratory and provide enough information to determine whether or not the sample is infected with avian influenza. Virus cultivation requires an average of 2-10 days to give a definite outcome, including more specific viral details.

The time taken to send the samples/results to and back from the laboratory will usually take longer than the actual test itself and possibly hinder treatment. For antiviral drugs to be effective, they must be consumed within the first 48 hours of symptom onset.

Rapid antigen detection tests are also available and only take 15-30 min on average, though only confirm the presence of influenza A viruses and fail to identify the subtype/strain of virus. Because seasonal influenza is also an influenza A virus, the tests cannot distinguish between a normal flu patient and a bird flu patient.

When Tests are Necessary

The symptoms of bird flu can in many ways be quite similar to that of the common seasonal flu since both are triggered by influenza viruses. However, aside from assessing how closely a patient's symptoms correspond to those of avian influenza, particular emphasis is also placed on a person's direct contact with either domesticated bird species or suspected bird flu patients during the previous 10 days.

A high fever above 38°C accompanied by any of the various symptoms characteristic to influenza usually only requires laboratory testing if there is considerable likelihood of virus contraction from recent contact with poultry or bird flu victims. Bird-to-human virus infection is in reality, relatively rare considering the mere 200+ cases that have occurred in humans and the millions of birds that have fallen victim to avian influenza.

Hospitalized patients who display radiographically confirmed pneumonia, acute respiratory distress syndrome (ARDS), or other severe respiratory illness for which an alternative diagnosis has not been established* and have made recent travels to known bird-flu-infected areas, are required in the United States to undergo an avian influenza test for H5N1, a highly pathogenic subtype of bird flu that often causes pneumonia and other severe symptoms.

*From CDC: Outbreaks of avian influenza A (H5N1) in Asia and interim recommendations for evaluation and reporting of suspected cases, United States, 2004

Developing an Instant Test

A November 2005 press release by the University of Colorado has announced the development of a new 'chip' test that potentially diagnoses 11 different strains of avian influenza four times faster than the current diagnosis of seasonal flu. Although still a little short of perfection, this new invention may one day allow doctors to instantaneously identify bird flu patients and administer treatment within a 48 hour period.

*Update* - The 'FluChip' was completed in August 2006 and currently enables a less than 12 hour-diagnosis of bird flu viruses. It has the ability to identify the specific influenza strain that has caused infection and will help increase the chances of victims to receive timely treatment. This test may prove particularly useful in situations where ordinary diagnosis procedures such as PCR or viral culture are unavailable due to a lack of facilities or time restraints. The test is currently being developed to further improve accuracy and is likely to be a key player in the battle against a global pandemic.

Bird Flu Diagnosis in Birds

Mucus samples or swabs from the cloacae of domestic poultry are usually used to test for the infection of bird flu in sick birds. In dead poultry, blood from the heart and brain are also collected for analysis and the general number of recommended samples from a poultry population is 6 - three samples from sick birds and three from birds that have recently died.

Once sent to the laboratory, these samples may either undergo PCR testing or viral antigen testing (see Diagnosing Human Cases) if rapid results are desired. An alternative and also quite common method of testing however, is a form of viral culture where the collected samples are implanted into 9-11 day old chicken eggs, so as to allow any potential viruses to develop. Allantoic fluid from the eggs can then be collected and further analysed by means of haemagglutinin tests to identify the subtype of bird flu present (See Subtypes).

To determine the pathogenicity of a particular virus subtype, 4-8 week old chickens are allowed to contract the disease and develop symptoms at their natural pace. The survival rate, illness and symptoms of the chickens over a ten-day period, beginning from the day of contraction, are recorded and used to assess the pathogenicity of the disease.

Poultry: Comparing Bird Flu to other Avian Diseases

There are several other diseases common to bird species that share similar symptoms to bird flu once poultry become infected. These may be mistaken for avian influenza (or vice versa) and include:

• Velogenic Newcastle Disease
• Acute Fowl Cholera
• Infectious Laryngotracheitis
• Infectious Bronchitis

As an example, the initial bird flu outbreaks in Indonesia in October 2003 were mistakenly diagnosed as Newcastle disease, resulting in a colossal loss of chicken population before the government finally announced that bird flu was the real culprit in January the following year. For such reasons, care is always taken to distinguish between the diseases.