IDENTIFICATION OF MUTATIONS IN THE INFLUENZA A GENOME ASSOCIATED WITH ADAMANTANE AND NEURAMINIDASE INHIBITOR RESISTANCE DIRECTLY FROM CLINICAL SPECIMENS

Objective: To evaluate the ability of pyrosequencing performed directly on clinical specimens (CS) to detect specific point mutations in the influenza A genome known to confer resistance to the antivirals adamantane (amantadine and rimantadine) and oseltamivir, a neuraminidase inhibitor.

 Methods: A sampling of respiratory virus surveillance specimens from 2006 to 2008 positive for influenza A by RT-PCR was tested.  Viral RNA was extracted from CS using the Total Nucleic Acid kit with the MagNA Pure LC® instrument (Roche Diagnostics). RT-PCR was performed to amplify products encompassing the region of the M2 gene and the NA gene known to confer resistance to adamantanes and oseltamivir, respectively.  Pyrosequencing of the PCR products were performed according to the manufacturer’s protocol (Biotage AB) to identify a change at the S31N in the M2 gene, and at the H274Y in the NA gene.  CS tested resulting in either a “Passed” or “Check” pyrosequencing quality rating were determined acceptable.  Any CS with a “Failed” pyrosequencing quality rating was determined unacceptable.

Results: Over the past two influenza seasons to date (2006-2008), 207 CS positive for influenza A virus by RT-PCR were tested for the S31N mutation by pyrosequencing.  Of those, 17 (8.2%) were determined unacceptable. 11 of 17 (64.7%) had a real-time RT-PCR crossing threshold (Ct) of >26.0.  Over the previous influenza season (2007-2008), 43 CS positive for influenza A(H1N1) virus were tested for the H274Y mutation in the NA gene.  Of those, 4 (11.7%) were unacceptable and all had a Ct value >26. Of all influenza A detected in CS from the 2006-2007 season in Wisconsin, the majority 376 out of 443 (84.9%) had Ct values ≤26.0. Of the 33A/H1 and 7 A/H3 CS tested during the 2007-2008 respiratory virus season, 1 A/H1 (3.0%) and 7 (100%) A/H3 were determined to be resistant to the adamantanes.

Conclusion: Previous studies (ASM abstract C71, 2007) indicated that direct pyrosequencing of CS using a Ct threshold of ≤26.0 was comparable to testing from cell culture isolates.  The results of this study confirm that pyrosequencing for resistance mutations from CS, using a Ct threshold of ≤26.0, is applicable for the majority of CS positive for influenza A.  Antiviral resistance mutation can be detected directly from CS in one day compared to up to seven days from cell culture isolates.  With emergence of antiviral resistance, this rapid testing has both clinical and public health implications for determining appropriate and timely antiviral treatment, especially in outbreak settings or with emergence of novel influenza strains.