H7N9: Lab Tests May Not Uncover Drug-Resistant Strains

Troy Brown

July 18, 2013

A characterization of the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 contained a mixture of drug-sensitive and drug-resistant strains, despite appearing to be sensitive to neuraminidase (NA) inhibitors in standard clinical tests.

Hui Ling Yen, PhD, from the Centre of Influenza Research, School of Public Health, Li Ka Shing Faculty of Medicine at the University of Hong Kong, and colleagues report their findings in an article published online July 16 in mBio.

The novel H7N9 viruses had previously been found to be resistant to M2 ion channel blockers, and therefore NA inhibitors such as oseltamivir and zanamivir are currently the frontline drugs for treating H7N9 infections.

During the first 3 months after the virus was initially reported in April 2013, more than 130 cases occurred. Most patients quickly developed pneumonia and acute respiratory distress syndrome, and 25% of them died. Clusters of human H7N9 infection occurred, and human-to-human transmission could not be ruled out in some of those instances, the authors write.

When Dr. Yen and colleagues used clonal sequencing to study the A/Shanghai/1/2013 isolate, they found it contained a mix of R (65%; 15/23 clones) and K (35%; 8/23 clones) at NA residue. However, the viral isolate, with its mix of R/K at residue 292, appeared sensitive to zanamivir and oseltamivir carboxylate in standard neuraminidase enzyme-inhibition assays.

When the researchers tested sensitivity using plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones), sensitivity to zanamivir was decreased by more than 30-fold and sensitivity to oseltamivir carboxylate was decreased by more than 100-fold compared with its plaque-purified wild-type counterpart with dominant R292 (93%, 14/15 clones).

When the researchers tested the plaque-purified A/Shanghai/1/2013-NAK292 virus in Madin-Darby canine kidney cells under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 - 1000 µM), viral titer was not reduced. In contrast, replication of the plaque-purified A/Shanghai/1/2013-NAR292 was completely inhibited at 250 µM oseltamivir carboxylate, and replication of the A/Shanghai/2/2013 was completely inhibited at 31.25 µM oseltamivir carboxylate.

Resistant strains of H7N9 can proliferate in patients treated with oseltamivir and zanamivir and promote their spread, corresponding author Robert Webster, PhD, from St. Jude Children's Research Hospital in Memphis, Tennessee, notes in a press release from the American Society for Microbiology.

"The clinical sample containing a mixed population of R/K may exhibit a phenotype that is sensitive to NA inhibitors, especially when phenotypic enzyme-based NA inhibition assays are used. It is therefore recommended that the emergence of the R292K mutation in H7N9 patients undergoing NA inhibitor treatment should be monitored using genotypic methods in combination with the phenotypic characterization of the virus," the authors conclude.

This study was supported by the Area of Excellence Scheme of the University Grants Committee in Hong Kong SAR and a contract from the US National Institute of Allergy and Infectious Diseases at the National Institutes of Health. The Chinese Centre for Disease Control and Prevention provided the human H7N9 isolates. The authors have disclosed no relevant financial relationships.

mBio. Published online July 16, 2013. Full text

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