Improved Molecular Tools Detect Influenza Virus

A set of molecular assays have been designed and evaluated for influenza viral infections that can produce results in one day without the need for additional equipment.

The molecular assays are sensitive and a good alternative for conventional diagnostic methods as they allow for the quantitative and qualitative detection of influenza virus and drug resistance mutations.

Scientists at the Erasmus University (Rotterdam, The Netherlands) designed validated and evaluated a set of real-time polymerase chain reaction (RT-PCR) assays for quantification and subtyping of human influenza A and B viruses from patient respiratory material, as well as four assays for detecting drug resistant mutations.

The scientific team analyzed 245 respiratory specimens from 87 patients living in Asia, Europe, and the United States of America, who were enrolled in a prospective study of influenza illness, including assessment of neuraminidase resistance. In addition, 96 prepandemic influenza A/H1N1 viruses from the epidemic of 2007-2008 were analyzed by the H275Y assay to check the robustness of the assay.

The influenza quantification assay was used to check for virus positivity and to obtain virus particle counts for all analyzed samples. Influenza A viruses were then subtyped and tested for presence of oseltamivir resistance mutations using the resistance RT-PCR assays. In total, 129 respiratory specimens tested positive for influenza A and 60 for influenza B virus. One sample tested positive for both virus types.

Although infection from H7N9, the new potential pandemic influenza strain, or H5N1, a continuing pandemic threat since 1997, can be identified by exclusion with a positive in the influenza matrix RT-PCR, but negative in RT-PCR typing. Development of rapid typing RT-PCR for these potential pandemic viruses may be useful in complementing the existing set. Within one working day, information can be obtained in parallel regarding influenza virus type or subtype, viral load, and antiviral susceptibility.

Martin Schutten, PhD, the lead author of the study said, “RT-PCR based assays have become the standard in most diagnostic laboratories worldwide in recent years. Our assays cover all currently circulating human influenza viruses and can detect major resistance mutations to oseltamivir. By introducing external quantification and internal standards, longitudinal assay performance can be monitored carefully and a virus particle count can be assigned to an analyzed sample.” The study was published in the May 2013 edition of the Journal of Molecular Diagnostics.

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