I T Prud'homme1, O Zoueva, J M Weber. 1. National Laboratory for Special Pathogens, Surveillance, Influenza and Viral Exanthemata Laboratory, Health Canada, Tunney's Pasture, Ottawa.
Abstract
BACKGROUND: Influenza A infections are an important cause of morbidity and mortality in the elderly and patients affected by chronic diseases or immunodeficiencies. Treatment and prevention of infection in hospitals and nursing homes often involve the use of amantadine, but resistant viruses may arise. OBJECTIVES: To assess the effectiveness of specific and sensitive methods for rapid screening and sequence confirmation of amantadine resistance, and the occurrence of amantadine resistance in recent influenza A virus isolates in Canada. STUDY DESIGN: A chicken antiserum-based enzyme linked immunoassay (ELISA) was developed and used to screen fifty influenza A isolates for amantadine resistance. Drug sensitivity was expressed as a percentage of virus growth inhibition. The efficiency of the assay was compared to that of a monoclonal antibody (mab)-based ELISA using influenza A strains from 1968 to 1994. Specific PCR primers, generated to amplify the M2 gene region where amantadine resistance mutations occur, were tested over a wide range of strains. Direct sequencing of the PCR fragments was performed to confirm the presence of resistance mutations. RESULTS: The polyclonal antiserum-based ELISA detected antigens from all recent H1N1 strains and H3N2 strains tested at an inoculum dilution ten-fold lower than the mab-based ELISA. Primers for the detection of amantadine resistance mutations consistently amplified a wide range of strains. The direct sequencing of the RT-PCR amplicons generated, detected resistance mutations in reassortant and control viruses, the only strains found resistant by ELISA. All influenza A isolates (H3N2, H1N1) tested, except resistant controls and two reassortant viruses, were amantadine-sensitive as indicated by greater than 50% virus growth inhibition. CONCLUSIONS: Influenza A virus susceptibility to amantadine could be detected by using an antiserum-based ELISA, offering a simple and more sensitive alternative to the mab-based assay. Coupled with direct sequencing of the M2 gene, it provides a reliable way to detect and confirm resistance in influenza isolates. However, no resistant clinical isolates were detected in the sample.
BACKGROUND:Influenza A infections are an important cause of morbidity and mortality in the elderly and patients affected by chronic diseases or immunodeficiencies. Treatment and prevention of infection in hospitals and nursing homes often involve the use of amantadine, but resistant viruses may arise. OBJECTIVES: To assess the effectiveness of specific and sensitive methods for rapid screening and sequence confirmation of amantadine resistance, and the occurrence of amantadine resistance in recent influenza A virus isolates in Canada. STUDY DESIGN: A chicken antiserum-based enzyme linked immunoassay (ELISA) was developed and used to screen fifty influenza A isolates for amantadine resistance. Drug sensitivity was expressed as a percentage of virus growth inhibition. The efficiency of the assay was compared to that of a monoclonal antibody (mab)-based ELISA using influenza A strains from 1968 to 1994. Specific PCR primers, generated to amplify the M2 gene region where amantadine resistance mutations occur, were tested over a wide range of strains. Direct sequencing of the PCR fragments was performed to confirm the presence of resistance mutations. RESULTS: The polyclonal antiserum-based ELISA detected antigens from all recent H1N1 strains and H3N2 strains tested at an inoculum dilution ten-fold lower than the mab-based ELISA. Primers for the detection of amantadine resistance mutations consistently amplified a wide range of strains. The direct sequencing of the RT-PCR amplicons generated, detected resistance mutations in reassortant and control viruses, the only strains found resistant by ELISA. All influenza A isolates (H3N2, H1N1) tested, except resistant controls and two reassortant viruses, were amantadine-sensitive as indicated by greater than 50% virus growth inhibition. CONCLUSIONS:Influenza A virus susceptibility to amantadine could be detected by using an antiserum-based ELISA, offering a simple and more sensitive alternative to the mab-based assay. Coupled with direct sequencing of the M2 gene, it provides a reliable way to detect and confirm resistance in influenza isolates. However, no resistant clinical isolates were detected in the sample.