Prolonged Shedding of Amantadine-Resistant Influenza A Viruses by Immunodeficient Patients: Detection by Polymerase Chain Reaction-Restriction Analysis

Abstract

Consecutive A (H3N2) influenza virus isolates from 2 influenza virus-infected immunodeficient patients treated with amantadine were examined using a novel polymerase chain reaction (PCR)-restriction analysis for resistance to this antiviral compound. The data indicate that immunodeficient patients may shed resistant viruses for prolonged periods and with different drug resistance mutations present at different times. This PCR-restriction technique allows rapid detection of amantadine- or rimantadine-resistant strains.

Incidence of Amantadine‐Resistant Influenza A Viruses in Sentinel Surveillance Sites and Nursing Homes in Niigata, Japan

“…Furthermore, it was revealed that the predominant amino acid substitution in the protein of resistant viruses was Ser-31-Asp in vitro and also in clinical samples, accounting for approximately 80% and suggesting it to be a reliable marker for viral resistance to amantadine and rimantadine (1,5,12,24). This information may be used to the best advantage to develop a rapid detection system for resistant strains by combining the high sensitivity of reverse transcription-polymerase-chain reaction technique and the high specificity of restriction analysis (13). We are now developing an approach for direct assessment of swab samples, using a modification of PCR-restriction analysis.…”

“…The antiviral agent amantadine specifically protects against influenza A virus infection, inhibiting the uncoating process of the virus after it has entered the host cell by endocytosis (1,8,11,13,18,22,23). Normally, acidification of the endosomal environment leads to a flow of protons to the inner part of the virion through ion channels formed by homotetramers of the M2 protein, a transmembrane protein.…”

An Off-Seasonal Amantadine-Resistant H3N2 Influenza Outbreak in Japan

“…Reverse transcription (RT) to create complementary DNA (cDNA) was performed using an influenza A generic primer, Uni12, as reported elsewhere (Hoffmann et al 2001). First, a nested PCR was performed using specific primers to amplify the M2 region, segment 7, of influenza A, as described previously (Masuda et al 2000) and then the PCR products were sequenced to examine mutations at positions 26, 27, 30, 31 and 34 in the transmembrane region of the M2 protein, conferring amantadine resistance (Klimov et al 1995). The templates were labeled by cycle sequencing reactions with fluorescent dye terminators (BigDye Terminator v 3.1 Cycle Sequencing Kit, Applied Biosystems), and the products were analyzed using an ABI 310 (Applied Biosystems) automatic sequencer following the manufacturer's instructions.…”

Influenza Infection in Patients Before and After Liver Transplantation

“…Patients who are immunodepressed may develop amantadineor rimantadine-resistant viruses associated with prolonged viral shedding. 2,96 Resistance to zanamivir in an immunocompromised child with influenza B has recently been described. 97 In a similar patient, an antigenic drift variant of influenza A similar to that occurring in the community has been described caused by prolonged viral replication.…”