WHO updates guidelines for labs dealing with influenza A virus

Highly pathogenic avian influenza (HPAI) caused by certain subtypes of influenza A virus in animal populations, particularly chickens, poses a continuing global human public health risk.

Direct human infection by an avian influenza A (H5N1) virus was first recognised during the 1997 outbreak in Hong Kong special administrative region of China. Subsequently, human infections with avian strains of the H9 and H7 subtypes have been further documented.

According to the World Health Organisation (WHO), the current outbreak in humans of avian A (H5N1) and the apparent endemicity of this subtype in the poultry in southeast Asia require increased attention to the need for rapid diagnostic capacity for non-typical influenza infections.

Laboratory identification of human influenza A virus infections is commonly carried out by direct antigen detection, isolation in cell culture, or detection of influenza-specific RNA by reverse transcriptase-polymerase chain reaction (PCR).

The optimal specimen for influenza A virus detection is a nasopharyngeal aspirate obtained within three days of the onset of symptoms, although nasopharyngeal swabs and other specimens can also be used. All manipulation of specimens and diagnostic testing should be carried out following standard biosafety guidelines outlined on the WHO's website (http://www.who.int/). 

The strategy for initial laboratory testing of each specimen should be to diagnose influenza A virus infection rapidly and exclude other common viral respiratory infections. Results should ideally be available within 24 hours.

Diagnostic procedure

Assays available for the diagnosis of influenza A virus infections include:

  • Rapid antigen detection. Results can be obtained in 15–30minutes. These include: near-patient tests for influenza that are commercially available; immunofluorescence assay, a widely used, sensitive method for diagnosis of influenza A and B virus infections; and enzyme immunoassay for influenza A nucleoprotein.
  • Virus culture. Provides results in 2–10days. Both shell-vial and standard cell-culture methods may be used to detect clinically important respiratory viruses. Positive influenza cultures may or may not exhibit cytopathic effects but virus identification by immunofluorescence of cell cultures or haemagglutination-inhibition (HI) assay of cell culture medium (supernatant) is required.
  • Polymerase chain reaction (PCR) and real time PCR assays. Primer sets specific for the haemagglutinin (HA) gene of currently circulating influenza A/H1, A/H3 and B viruses are becoming more widely used. Results can be available within a few hours from either clinical swabs or infected cell cultures. Additionally, several WHO collaborating centres are developing PCR and RT-PCR reagents for non-typical avian/human influenza strains.


Any specimen with a positive result using the above approaches for influenza A virus and suspected of avian influenza infection should be further tested and verified by a designated WHO H5 reference laboratory.

Laboratories that lack the capacity to perform specific influenza A subtype identification procedures are requested to forward specimens or virus isolates to a national influenza centre or WHO H5 reference laboratory, and to inform the WHO office in that country that such an action has been taken.

Influenza A subtypes

Immunofluorescence assay (IFA) can be used for the detection of virus in either clinical specimens or cell cultures. Clinical specimens, obtained as soon as possible after the onset of symptoms, are preferable as the number of infected cells present decreases during the course of infection.

Performing IFA on inoculated cell cultures is preferable as it allows for the amplification of any virus present. The materials required include the WHO Influenza Reagent Kit for the Identification of Influenza A/H5 Virus (1997-98, 2003 or 2004 version).

Specific staining should be an intense intracellular apple-green fluorescence. Nuclear and/or cytoplasmic fluorescence may be observed. It is important to ensure that cell density is adequate. One or more intact cells showing specific intracellular fluorescence can be accepted as a positive result.

Because commercially available monoclonal antibodies for the subtyping of influenza A/H1 have been shown to cross-react with influenza A/H5 subtypes, including current (2004) strains, confirmatory testing should be carried out using the monoclonal antibody provided in the WHO kit.

Virus culture

Virus isolation is a sensitive technique with the advantage that virus is available both for identification and for further antigenic and genetic characterisation, drug susceptibility testing, and vaccine preparation. MDCK cells are the preferred cell line for culturing influenza viruses.

Identification of an unknown influenza virus can be carried out by IFA using specific monoclonal antibodies or, alternatively, by haemagglutination (HA) and antigenic analysis (subtyping) by haemagglutinationinhibition (HAI) using selected reference antisera. Unlike other influenza A strains, influenza A/H5 will also grow in other common cell lines such as Hep-2 and RD cells.

Standard biosafety precautions should be taken when handling specimens and cell cultures suspected of containing highly pathogenic avian influenza A. The golden rule, says the WHO, is that clinical specimens from humans and from swine or birds should never be processed in the same laboratory.

In terms of results interpretation, the highest dilution of virus that causes complete haemagglutination is considered to be the HA titration end-point. The HAI end-point is the last dilution of antiserum that completely inhibits haemagglutination. The titre is expressed as the reciprocal of the last dilution.

Identification of the field isolate is carried out by comparing the results of the unknown isolate with those of the antigen control. An isolate is identified as a specific influenza A subtype if the subtype-specific HAI titre is four-fold or greater than the titre obtained with the other antiserum. Nonspecific agglutinins may be present in sera and may result in false-negative reactions; alternatively, some isolates may be highly sensitive to non-specific inhibitors in sera, resulting in false-positive reactions.

PCR

PCR is a powerful technique for the identification of influenza virus genomes. The influenza virus genome is single-stranded RNA, and a DNA copy (cDNA) must be synthesised first using a reverse transcriptase (RT) polymerase.

The procedure for amplifying the RNA genome (RT-PCR) requires a pair of oligonucleotide primers. These primer pairs are designed on the basis of the known HA sequence of influenza A subtypes and of N1 and will specifically amplify RNA of only one subtype. DNAs generated by using subtype-specific primers can be further analysed by molecular genetic techniques such as sequencing.

The expected size of PCR products for influenza A/H5 is 219bp, for A/H9 is 383bp, and for N1 is 616bp. If the test is run without a positive control, products should be confirmed by sequencing and comparison with sequences in deposited databases. The absence of the correct PCR products – a negative result – does not rule out the presence of influenza virus. Results should be interpreted together with the available clinical and epidemiological information. Specimens from patients with a high probability of infection with influenza A/H5 or H9 should be tested by other methods such as IFA, virus culture or serology to rule out influenza A (A/H5 or H9) infection.

Lab confirmation

All laboratory results for influenza A/H5, H7 or H9 during interpandemic and pandemic alert periods of the WHO Global Influenza Preparedness Plan should be confirmed by a WHOH5 reference laboratory or by a WHO recommended laboratory. Influenza A/H5, H7 or H9-positive materials, including human specimens, RNA extracts from human specimens, and influenza A/H5, H7 or H9 virus in cell-culture fluid or egg allantoic fluid, should be forwarded to a WHOH5 reference laboratory or a WHO recommended laboratory.

Finally, the WHO concludes that communication and publication of analysis results should be according to its own guidance for the timely sharing of influenza viruses/specimens with potential to cause human influenza pandemics.

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