rapid and sensitive method for the diagnosis of the "avian flu" H5 sub-type of influenza A virus

ABSTRACT

Avian influenza, an infectious disease of birds, is caused by type A strain of influenza virus. Within the first 3 months of 2006. 46 human cases of avian influenza in humans worldwide were found where of 31 have died with a mortality rate of about 67%. Therefore, the need for a rapid and sensitive method to diagnose the infection, subtype, and quantify the virus is of paramount importance in the management of this pandemic. Virology unit, Kuwait University, Mubarak hospital in Kuwait. 80 patients with lower respiratory tract infections. We have developed a sensitive and specific multiplex RT-PCR capable of detecting common nine respiratory viruses causing acute respiratory disease in Kuwait. Those found positive for influenza A virus, were then subjected to a quantitative Real-Time PCR to sub-type the virus as H5 and to measure the amount of virus in respiratory samples. A 189 bp fragment of the influenza virus A H5 gene was amplified with specific primers and quantitatively detected with probes supplied in the LightMix [TIB MOLBIOL, Berlin]. The supplied standard dilutions of influenza A H5 virus cDNA ranging from 101 to 106 copies/reaction allows the absolute quantification of the viral cDNA in the unknown samples. A standard curve using standard increasing dilutions of influenza A H5 ranging from 101 to 106 cDNA copies /ml was plotted to quantitate influenza A virus of the H5 subtype. The minimum detection limit was 10 copies/ml. Specificity of the primer/probe mixture was tested on samples containing rhino-, corona-, RSV, measles and mumps viruses. Out of the 10 of 80 [12.5%] patients with acute respiratory disease who were influenza virus A positive, none was found to be of the H5 sub-type of influenza A virus so far in Kuwait. The combination of the multiplex RT-PCR developed in our laboratory and the real time PCR provides a fast easy, specific, and accurate system for the detection of influenza A H 5 subtype