the novel swine origin influenza a virus ( s - oiv ) ( h1n1 ) caused pandemic in 2009 - 2010 . oseltamivir ( tamiflu , hoffmann laroche ) is recommended for the treatment of s - oiv infection because it is naturally resistant to amantadine and rimantadine but is susceptible to the neuraminidase inhibitors , oseltamivir , and zanamivir ( relenza , glaxo smithkline ) although the oseltamivir - resistant neuraminidase ( na ) h275y ( n1 numbering ) was detected in some isolates [ 24 ] . here , we precisely report the virus shedding of the pandemic s - oiv after oseltamivir treatment ( table 1 ) . a 24-year - old female in shanghai , china was infected with the pandemic s - oiv in september 2009 . she had received a seasonal flu vaccination ( sanofi pasteur ) the previous year . her first symptom was a sore throat , and on the 4th day after the onset of her sore throat , she had a high fever ( > 38.5c ) . the next day ( the 5th day after onset ) , she visited a clinic . oseltamivir ( 75  mg  2 ) was administered at 36 hours after the onset of her high fever for five days . on the second day of oseltamivir administration , her high fever disappeared , and all eight segments of the s - oiv genome were cloned from her pharyngeal swabs , and the sequences of the pandemic s - oiv were confirmed . since then , pharyngeal swabs were collected everyday for eight days and subjected to virus isolation and examined for virus genome rna ( from the 6th to the 13th day ) . each pharyngeal swab was aliquoted into three fractions : for virus isolation , for rt - pcr , and for real - time rt - pcr , and each experiment was performed in a different lab . for virus genome detection , rna was extracted from the pharyngeal swabs using trizol ( invitrogen ) . the rna was then analyzed by real time rt - pcr for s - oiv haemagglutinin ( ha ) , which was performed in a smartcycler ii system ( cepheid ) using a quantitect probe rt - pcr kit ( qiagen ) with the primers ( ha primer 1 and 2 ) and ha probe listed in table 2 . at the same time , the rna was analyzed for the eight genome segments by rt - pcr . the cdna was reverse transcribed by mmlv rtase ( takara ) using uni12 ( agcaaagcagg and agcgaaagcagg ) , before being subjected to pcr using la - taq ( takara ) for the coding frames of the eight genome segments and the primers listed in table 2 . her pharyngeal swabs were also inoculated into mdck cells after filtration through 0.22  m filter ( millipore ) and then incubated in dmem with 2  g / ml trypsin ( difco ) , 1  mg / ml bsa ( sigma ) , 50  units / ml of penicillin ( invitrogen ) , 50  g / ml of streptomycin ( invitrogen ) , and 0.1  mg / ml kanamycin ( invitrogen ) at 37c for the detection of cytopathic effects ( cpes ) . when cpes were detected , the pandemic s - oiv genome segments were also identified by rt - pcr of the culture supernatant . oseltamivir is accepted as the standard treatment for s - oiv [ 2 , 4 ] . although oseltamivir was effective , the virus was isolated from her pharyngeal swabs on the second ( 20 hours ) and third ( 44 hours ) days of oseltamivir administration . the eight viral genome segments were detected by rt - pcr , and ha genome was detected by real - time rt - pcr until the day after the end of oseltamivir administration . no other people around her showed any symptoms .   by 36 hours of oseltamivir administration in previous cases , the influenza a / texas/91 ( h1n1 ) virus had disappeared from the patients ' nasopharyngeal swabs . in this case , the virus ( influenza virus a / shanghai / p1/2009 ( h1n1 ) ) was still isolated from the patient 's pharyngeal swab on the third day ( 44 hours ) of oseltamivir administration , and the viral genome segments were detected until the day after oseltamivir administration was stopped . the + + + and + + scores of real - time rt - pcr correlated with those of virus isolation . the prolonged detection of viral rna without the oseltamivir - resistant mutation of na by rt - pcr or real - time rt - pcr from the s - oiv - infected patients administrated with oseltamivir was also reported previously [ 8 , 9 ] . all of these reports indicate that s - oiv could persist in the patients although shedding of s - oiv may vary from case to case . however , due to their high sensitivity , rt - pcr and real - time rt - pcr may cause misdiagnosis , and the classical virus isolation and clinical symptoms are still recommended . the classical virus isolation and clinical symptoms are recommended for the evaluation of oseltamivir treatment although rt - pcr and real - time rt - pcr are convenient . written informed consent was obtained from the patient for publication of this case report and any accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal . l. weng and q. wang collected samples and performed rt - pcr , w. wang , p. ren , and v. deubel performed virus isolation and real - time rt - pcr , and l. weng and t. toyoda wrote the paper .
we analyzed the virus shedding of an oseltamivir - treated patient who had been infected with the pandemic swine - origin influenza a ( h1n1 ) virus which had an oseltamivir - sensitive neuraminidase . 
 the virus was isolated from the pharyngeal swabs of the patient using mdck cells , and the virus genome rna was detected in the same samples both by real - time rt - pcr and rt - pcr . 
 the virus was isolated until 44 h after oseltamivir administration although the virus genome was detected until one day after oseltamivir treatment was stopped . due to their high sensitivity , rt - pcr and real - time rt - pcr may cause misdiagnosis by detection of viral genome which does not infect , and classical virus isolation and clinical symptoms are recommended for the evaluation of oseltamivir treatment .