Vaccine and Infectious Disease Division

Tracking down resistant strains

H1N1 virus

An image of novel H1N1 taken in the laboratories of the Centers for Disease Control and Prevention, Atlanta.

VIDI scientists are working to develop new techniques to rapidly detect strains of novel H1N1 with resistance to oseltamivir, the common antiviral drug.

Resistance to oseltamivir, widely known by its brand name Tamiflu, has been widespread and problematic in seasonal flu in the past, including the 2008 flu season, in which nearly all circulating strains were oseltamivir-resistant.  Resistant novel H1N1 is rare (only approximately 1 percent of cases tested by the Centers for Disease Control and Prevention are oseltamivir-resistant), but researchers and clinicians want to prepare for the ability to detect large numbers of resistant strains during the 2009-2010 flu season.

Visiting VIDI fellow Dr. Christian Renaud, along with VIDI staff scientist Dr. Jane Kuypers and VIDI co-director Dr. Larry Corey in VIDI’s molecular diagnostic laboratory, are developing in-house techniques to detect the single base pair change in pandemic H1N1 that is the most common known oseltamivir-resistant mutation.  This mutation (specifically, a change of one amino acid, H275 to Y, in the neuraminidase gene of the virus) is the same one found in the 2008 oseltamivir-resistant seasonal flu strain.

“Because of the large immunosuppressed population here at the Seattle Cancer Care Alliance, we wanted to have a test to detect the mutation in every patient,” Renaud said. “This is not yet being done by any other lab that I know of.”

Although researchers still don’t know whether a oseltamivir-resistant strain of novel H1N1 could reach the same levels as the 2008 resistant seasonal flu strain, certain populations may be more susceptible to developing resistant strains, including children and immunosuppressed patients who tend to shed virus for a long time and are thus remain on medication for longer, and those who take oseltamivir prophylactically.  In unrelated cases, two transplant patients at the SCCA were found to have resistant strains of novel H1N1 in early summer 2009.

Right now, the CDC and other labs use a technique called the Neuraminidase activity inhibition assay to detect antiviral-resistant strains of novel H1N1.  The technique works very well, Renaud said, but it is slow and therefore mainly used for epidemiological purposes, not diagnoses or treatment decisions.  Other established techniques for detecting resistant flu strains, of which a type of sequencing called pyrosequencing is one of the most common, are similarly too expensive or too time consuming to perform in large quantities, Renaud said.  So he and his colleagues have to find a new way to be able to screen all immunosuppressed patients at the SCCA.

To establish such a technology, Renaud and Kuypers are exploring several options.   One possibility is a type of ligation assay, where first H1N1 RNA is reverse transcribed to DNA and then short synthetic DNA sequences, or probes, are made to specifically stick to the regions of the novel H1N1 DNA surrounding the mutation.  An enzyme is then used that will attach the two probes together only if the oseltamivir-resistant mutation is present. The probes are labeled in such a way that they can be detected if they are attached.  This method is specific and sensitive, Renaud said, and can be run on many patients at once, but is slightly time consuming.

Another technology they are pursuing uses a new machine in the molecular diagnostic laboratory called a Luminex 200 analyzer, which would work similarly to the ligation assay but can run many different tests at once, so the team could use it to screen not only for oseltamivir-resistant novel H1N1, but for other types of flu or respiratory viruses as well as detecting other mutations in flu viruses.

“That is really the final goal,” Renaud said.

The team hopes to have a working assay in place in the next few months to start screening SCCA patients this flu season.