Oseltamivir resistance in an influenza A (H3N2) virus isolated from an immunocompromised patient during the 2014–2015 influenza season in Alberta, Canada

This manuscript describes the identification of an oseltamivir‐resistant influenza A (H3N2) virus in a respiratory specimen collected from an immunocompromised patient in Alberta, Canada, during the 2014–2015 influenza season. Following treatment with oseltamivir, neuraminidase (NA) gene sequencing indicated the presence of an R292K mutation. Phenotypic susceptibility testing by the NA‐Star assay indicated a highly reduced inhibition by oseltamivir and normal inhibition by zanamivir. The use of zanamivir following identification of the oseltamivir‐resistant strain, combined with a partial immune reconstitution, was followed by a suggested decrease in the nasopharyngeal viral load in the nasopharynx and clinical improvement of the patient.


| METHODS
Nasopharyngeal (NP) specimens were taken at four time points ( Fig. 1). Pre-admission swabs from the start of symptoms were not available. Real-time reverse transcription PCRs were undertaken for influenza A matrix (M) gene detection and subtyping of H3 and pdm09 H1 viral targets. These were as per routine laboratory standard operational procedures on all NP specimens. Sequencing of the NA gene was undertaken as previously described. 4 Careful manual examination of the electropherogram at bases that code for amino acid 119-294 was performed to monitor for the presence of previously described single nucleotide polymorphisms (mixed or in total), described above as well as deletions.
For strain analysis from the primary specimen, hemagglutinin (HA) sequencing and alignment were done as previously described. [4][5][6] The NA-Star assay was performed according to manufacturer's recommendations. Two real-time RT-PCR-positive specimens were cultured on MDCK and MDCK-SIAT1 cells using previously pub- Clinical information was provided by the managing physicians.
As this was a case report, and was undertaken as part of normal surveillance activities and public health activities for the emergence of antiviral resistant strains of influenza, research ethics board clearance was not required by the University of Alberta, Research Ethics Office.
However, since the manuscript involved a detailed rereview of the patient record, a representative of the data custodian, Alberta Health, was engaged in the analysis.

| RESULTS
The patient was a 51-year-old male previously diagnosed with a stage IIA bulky diffuse large B-cell lymphoma in April 2011. On November F I G U R E 1 Laboratory test results and clinical progression of an immunocompromised patient infected with an oseltamivir-resistant influenza A (H3N2) virus. Arrows indicate periods of antiviral administration in relation to timeline. Solid blocks indicate key clinical presentations in relation to timeline. C t values for the influenza A virus M and H3 genes are listed at the bottom of the figure in relation to timelines and represent the four specimens collected. Hemagglutinin (HA) sequencing allowed the specimens to be subgrouped as a 3C.2a virus for three specimens, the fourth specimen having a viral load too weak to allow for HA sequencing. Neuraminidase sequencing indicated that the first NP swab collected contained a wild-type virus with R292 in N2. The second specimen collected contained a R292K mutation in N2 and had a highly reduced inhibition by oseltamivir (20884.78-fold above NI) but a normal inhibition by zanamivir (3.21-fold above NI). A decrease in viral load, as evidenced by increasing C t values for M and H3 RT-PCR, was noted for the third and fourth specimens following treatment with zanamivir, an improved immune status of the patient, and resolution of fevers and upper respiratory tract infection (URTI) symptoms. Abbreviations are as follows: autologous stem cell transplantation (ASCT), chemotherapy (CTx), cycle threshold (C t ), nasopharyngeal (NP), and reverse transcriptase polymerase chain reactions (RT-PCRs).

| DISCUSSION
This study describes the identification of an oseltamivir-resistant  The R292K mutation has been associated with decreased viral fitness in ferret models and it is possible that reconstitution of the patient's immune system would have at least partly cleared this strain. 10 The phenotypic susceptibility test data also suggest that the isolate from Day +5 post-ASCT would have had normal levels of inhibition if treated with zanamivir. From Fig. 1, zanamivir was introduced during a period of time when the patient was still strongly immunosuppressed. Genotyping of influenza strains has not always provided definitive evidence of reduced sensitivity to NA inhibitors in influenza A (H3N2) virus. The gold standard is still phenotypic resistance testing using a variety of well-published techniques. 11 However, questions still arise regarding the mutations generated in cell culture systems 12 which may lead to artifactual results. 13 There are also some concerns that the NA-Star assay, a chemiluminescent assay, may not be able to detect low-level increases in IC 50 within isolates containing the R292K polymorphism that were detectable when fluorescent assays were used. 14 The continued description and comparison of influenza A (H3N2) cases with resistance to NA inhibitors will lead to a better understanding of antiviral susceptibility, and inferences can be made from primary sequence analysis of specimens with less emphasis of phenotypic analysis from culture isolates.