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Respiratory Viruses Slide Set

Laboratory Diagnosis of Influenza Viruses Infection

E. Laboratory Diagnosis

During epidemics, a presumptive diagnosis can be made on the basis of the clinical symptoms. However, influenza A and B can co-circulate, and mixed infections of influenza and other viruses have been reported. Isolated cases of suspected influenza should be investigated for these may represent the first cases of an impending epidemic.

  1. Virus Isolation - Throat swabs, NPA and nasal washings may be used for virus isolation. It is reported that nasal washings are the best specimens for virus isolation. The specimen may be inoculated in embryonated eggs or tissue culture. 10-12 day embryonated eggs are used for virus isolation. The specimen is inoculated into the amniotic cavity. The virus replicates in the cells of the amniotic membrane and large quantities are released back into the amniotic fluid. After 2-3 days incubation, virus in the amniotic fluid can be detected by adding aliquots of harvested amniotic fluid to chick, guinea pig, or human erythrocytes. Pathological specimens can be inoculated on to tissue cultures of kidney, chicks or a variety of other species. Rhesus monkey cells are the most sensitive. Although no CPE is produced, newly produced virus can be recognized by haemadsorption using the cells in the tissue culture, and haemagglutination using the culture medium which contains free virus particles. Influenza B virus and occasionally influenza A will produce a CPE in MDCK cells. Influenza viruses isolated from embryonated eggs or tissue culture can be identified by serological or molecular methods. Influenza viruses can be recognized as A, B, or C types by the use of complement fixation tests against the soluble antigen. (A soluble antigen is found for all influenza A, B or C type virus but antibody against one type does not cross react with the soluble antigen of the other. The further classification of influenza isolates into subtypes and strains is a highly specialized responsibility of the WHO reference laboratories. The HA type is identified by HAI tests, the NA type is also identified.
  1. Rapid Diagnosis by Immunofluorescence - cells from pathological specimens may be examined for the presence of influenza A and B antigens by indirect immunofluorescence. Although many workers are convinced of the value of this technique, others have been disappointed with the specificity of the antisera and the level of background fluorescence that makes the test difficult to interpret. EIA tests for the detection of influenza A viral antigens are available that are easier to interpret than immunofluorescence. PCR assays for the detection of influenza RNA have also been developed but there usefulness in a clinical setting is highly questionable.
  2. Serology - Virus cannot be isolated from all cases of suspected infection. More commonly, the diagnosis is made retrospectively by the demonstration of a rise in serum antibody to the infecting virus. CFT is the most common method used using the type specific soluble antigen. However, the CF test is thought to have a low specificity. A more specific test is the HAI test. Infection by influenza viruses results in a rise in serum antibody titre, but the requirement for a 4-fold or greater rise in titre of HI of CF antibody reflects the inaccuracy of these tests for detecting smaller increases in antibody. A more precise method for measuring antibody is by SRH. SRH is more sensitive than CF or HAI tests and has a greater degree of precision. A 50% increase in zone area represents a rise in antibody and is evidence of recent infection. Sera do not have to be pretreated to remove non-specific inhibitors which plaque the HAI test. SRH may well replace CF and HAI tests in diagnostic laboratory in future.

F. Treatment

Influenza epidemics are responsible for massive disruption to industry, and for a significant number of deaths, particularly in the elderly and the very young. At present, treatment of influenza is entirely symptomatic. Salicylates should be avoided in children because of the link with Reye's syndrome. 2 compounds, amantidine and ribavirin, with antiviral activity against influenza have been identified and may be of value.

  1. Amantidine - this compound inhibit the growth of influenza viruses in cell culture and in experimental animals. Amantidine is only effective against influenza A, and some naturally occurring strains of influenza A are resistant to it. The mechanism of action of amantadine is not known. It is thought to act at the level of virus uncoating. The compound has been shown to have both therapeutic and prophylactic effects. Amantidine significantly reduced the duration of fever (51 hours as opposed to 74 hours) and illness. The compound also conferred 70% protection against influenza A when given prophylactically. Amantidine can occasionally induce mild neurological symptoms such as insomnia, loss of concentration and mental disorientation. However, these symptoms quickly developed in susceptible individuals and cease when treatment is stopped. The therapeutic and prophylactic activity of amantidine is now generally accepted and numerous analogues of this compound have been prepared. Rimantadine is not as effective as amantadine but is less toxic. Prophylaxis with 200mg of amantadine per day for 5 to 6 weeks or for the duration of the influenza A outbreak is not recommended for all persons. However, elderly persons with chronic underlying disease, institutionalized persons, staff and patients in hospital, close contacts of an index case, and patients who cannot receive influenza A vaccine due to sensitivity to egg protein may benefit from prophylaxis. Amantadine can also be used for therapy of uncomplicated influenza A infections. The recommended dose is 200mg for 5 days. Rimantadine may be used in place of amantadine for prophylaxis and the treatment of uncomplicated influenza A infections.
  2. Rimantidine - this compound is similar to amantidine but has fewer side effects. It is approved by the FDA for the treatment and prophylaxis of influenza A infection in persons one year or older. It should be used for uncomplicated influenza A infections only since it is thought to be less effective than amantidine. Amantadine and rimantadine resistant viruses are readily generated in the laboratory. Resistance has been linked to changes in the M2 protein. To date, the emergence of resistant influenza A has been documented primarily in young children undergoing therapy with rimantadine. The resistant viruses had been transmitted and caused influenza. The universal susceptibility of all types of naturally occuring influenza A isolated from man and animals suggests that resistance will be found only in individuals treated with the drug. The reason for the natural selection of the susceptible phenotype of influenza A in nature is not known.
  3. Zanamivir - the rational approach to drug design has led to the design of several potent inhibitors of influenza neuraminidase. Zanamivir was the first neuraminidase inhibitor available for clinical use and is effective against both influenza A and B. Because of its poor bioavailability, zanamivir must be administered by inhalation. Zanamivir had been shown to be effective and devoid of significant side effects in clinical trials. It is now approved by the FDA for use as treatment for influenza A and B in persons 12 years or older but not for prophylaxis.
  4. Oseltamivir - oseltamivir is another neuraminidase inhibitor but unlike zanamivir, it can be given orally. Like zanamivir, it had been shown to be effective and devoid of significant side effects in clinical trials. It is approved by the FDA for use as treatment for influenza A and B in persons 18 years or older. It is also approved for prophylaxis in persons 13 years or older. Its lack of side effects would make particularly attractive in a family setting although its higher cost compared to amantidine and rimantidine should be taken into account.

G. Prevention

Vaccines against influenza have been around for 50 years. Despite this, the efficacy of influenza vaccines is still questioned, and the ability of vaccines to limit epidemic infection has not been proven.

1. Immunity to Influenza - the results of challenge studies indicated that immunity is induced by the host responses to the virus haemagglutinin (HA) and to neuraminidase (NA). Antibody against HA is the most important component in the protection against influenza viruses. In addition to conferring relative protection against infection, serum HI is reported both to reduce the severity of infection and decrease virus spreading in infected persons. Serum anti-neuraminidase Ab has also been shown to contribute protection against influenza infection. A generally held view is that serum HI antibody is more important in determining immunity than anti-neuraminidase antibody. It is clear that an influenza vaccine must contain both HA and NA antigens in a form which will stimulate the production of neutralizing antibody, local IgA antibody and possibly cellular immunity.

2. Types of vaccine

  1. Whole virus vaccines - whole inactivated virus vaccines were the first influenza vaccines to be produced. The currently circulating strain of influenza is inoculated into embryonated eggs, harvested 2-3 days later and inactivated. this vaccine confers protection in 60-90% of vaccinees and the protection lasts for 1-5 years, depending on the vaccine strain and the age of the vaccinee. However, the subsequent infecting virus may show slow antigenic drift and the vaccine induced antibody will be less effective in conferring protection against the new strains.
  2. Split virus vaccines - Because of the high incidence of reactions seen in vaccinees given whole, inactivated virus vaccine, attempts have been made to produce a vaccine which is less reactogenic but conserving immunogenicity. Split vaccines were prepared inactivated particles disrupted with detergents. These vaccines have been shown to induce fewer side effects in the vaccinees and are just are immunogenic as whole virus vaccine. Whole virus vaccine should not be used in children.
  3. Subunit virus vaccines - subunit vaccines have been prepared which contained only the HA and NA antigens. These are used in aqueous suspension or may be absorbed to carriers such as alhydrogel. Volunteers given subunit vaccines experienced fewer reactions than those given whole virus vaccines and those given aqueous vaccine experienced fewer reactions than those given the absorbed subunit vaccine. therefore, the best vaccines available at present are the aqueous subunit vaccines, although some authorities have questioned the effectiveness of subunit vaccines.
  4. Live attenuated vaccines - there is experimental evidence that immunization with live, attenuated influenza virus vaccines induce a solid immunity than do inactivated vaccines. Normal methods for attenuation, such as repeated passages and temperature adaptation require a long period to complete, and probably too long for the vaccine to become available for immunization against the current influenza strain. To circumvent this problem , already attenuated strains have been mixed with wild-type virus to produce recombinants which contain the RNA fragments which code for wild-type HA and NA, and all the other genetic material form the attenuated strain. These recombinants can be produced relatively quickly. When given intranasally, produced few side effects.

Although research to develop live attenuated vaccines has been pursued for 20 years, basic problems remains particularly in the area of purification. The vaccine must also be shown to be attenuated and safe. It is estimated that if the safeguards are to be satisfied, 2 years would be needed for the development of an attenuated vaccine. This makes their development impractical , since by the time the vaccine virus can be made available, the epidemic strain against which the vaccine has been prepared would have disappeared. For an attenuated vaccine to be a practical proposition, the development time must be down to 6-9 months.

3. Recommendations - At present, no live attenuated vaccine is available for general use. The vaccines that are currently available are produced from virus grown in embryonated eggs. The aqueous subunit vaccine is the most acceptable formulation. These vaccines produce few reactions and confer protection in 60-90% of vaccinees. Vaccination is recommended for the elderly and individuals at risk for severe infection. In addition, key personnel n industry and social and medical services. It is highly debatable whether the vaccine should be given to the general population. Influenza immunization is strongly recommended for adults and children with any of the following:

1. Chronic respiratory disease
2. Chronic heart disease
3. Chronic renal failure
4. Diabetes mellitus and other endocrine disorders
5. Immunosuppresion due to disease or treatment

Immunization is also recommended for residents of nursing homes and old peoples' homes and other long stay facilities where rapid spread is likely to follow. Two types of vaccines are available in the UK; "split virus vaccines", and "surface antigen" vaccine which contains highly purified HA and NA antigens prepared from disrupted virus particles. Both vaccines are suitable for use in children.

4. WHO Influenza Surveillance Program

The WHO has a network of around 110 influenza centres worldwide that regularly submit new influenza isolates to the 4 WHO collaborating centres (US, UK, Japan, Australia) for analysis. The aim is to detect  new and potentially dangerous strains of influenza at the earliest moment so that measures can be enacted in the event of a pandemic. The strains used in current influenza vaccines are supplied to the vaccine by the WHO. It is normally a trivalent vaccine: one H3N2, one H1N1 and one influenza A sutype. The sutypes selected are those that are normally the most antigencally diverse strains considered to have to greatest epidemic potential.


H. The H5N1 avian influenza outbreak in Hong Kong 1997

In the latter half of 1997, an outbreak occurred in Hong Kong whereby 18 persons were infected by an avian influenza A, serotype H5N1. Of these 6 died, and 3 others were severely ill. The source of the outbreak was infected chickens and the outbreak stopped after all the chickens were slaughtered in the territory. Large-scale serological studies carried out showed that workers in the poultry industry were particularly at risk of infection although none complained of any symptoms. There was evidence of limited human to human transmission. It was postulated that the strain of avian influenza involved was unusually virulent; it had multiple basic amino acids near the cleavage site of the haemagglutinin protein, which as a result may render the haemagglutinin susceptible to a wider range of proteases. Since that outbreak, no more cases have occurred. In 1999, there were reports of human infections by avian influenza A H9N2 in Hong Kong and in Mainland China. However, all these cases were very mild and it is thought that the virus was unlikely to pose a large public health risk.

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Respiratory Viruses Slide Set