Development of cycling probe based real-time PCR methodology for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced baloxavir susceptibility

Baloxavir marboxil has been used for influenza treatment since March 2018 in Japan. After baloxavir treatment, the most frequently detected substitution is Ile38Thr polymerase acidic protein (PA/I38T), and this reduces susceptibility A viruses. To rapidly investigate frequency of PA/I38T (H1N1)pdm09 (H3N2) viruses clinical samples, we established a rapid real-time system to detect single nucleotide polymorphisms PA, using cycling probe PCR. We designed two sets probes that were labeled with either 6-carboxyfluorescein (FAM) or 6-carboxy-X-rhodamine (ROX) identify PA/I38 (wild type strain) PA/I38T, respectively. The PCR showed dynamic linear range 101 106 copies high sensitivity plasmid DNA controls. This discriminated between wild well. During 2018/19 season, 377 A-positive samples collected Japan before antiviral treatment. Using our system, no (0/129, 0.0%) substitutions four (4/229, 1.7%) prior In addition, found variant siblings who did not received during an infection caused by afflicted entire family. Although human-to-human transmission may have occurred closed environment, prevalence was still limited. Our probe-PCR thus useful surveillance possessing PA/I38T.