Article

Highly Sensitive and Quantitative Detection of the H274Y Oseltamivir Resistance Mutation in Seasonal A/H1N1 Influenza Virus

Wadsworth Center, New York State Department of Health, Albany, NY 12201-2002, USA.
Journal of clinical microbiology (Impact Factor: 4.23). 10/2010; 48(10):3517-24. DOI: 10.1128/JCM.01031-10
Source: PubMed

ABSTRACT A C-to-T transition mutation in the neuraminidase gene from seasonal A/H1N1 causes a His-to-Tyr mutation at amino acid position 275 (H274Y, universal N2 numbering), conferring resistance against oseltamivir (Tamiflu). This mutation was first detected in clinical samples in Europe during the 2007-2008 influenza season. Viruses with this mutation reached a prevalence of ∼11% by the end of the season in North American isolates tested by the CDC. We developed a highly sensitive and specific quantitative real-time reverse transcriptase PCR assay to detect the H274Y mutation. This assay utilizes a 5'-methyl-isocytosine (isoC) residue and fluorescent reporters on genotype-specific primers. During PCR, a quencher coupled to isoguanine (isoG) is site-specifically incorporated complementary to the isoC/dye, resulting in loss of fluorescence. Optimization of primers and assay conditions produced a limit of detection of 100 gene copies per reaction for both wild-type and H274Y genotypes. In samples with mixed populations, it can reliably detect as little as a 1% wild-type or 0.1% H274Y component. This high sensitivity makes the assay usable on samples with viral loads too low for dideoxy or pyrosequencing analysis. Additionally, the assay distinguishes seasonal A/H1N1 from A/H3N2, influenza B, or 2009 pandemic A/H1N1, making it useful for influenza virus subtyping as well as for drug resistance detection. We probed seasonal A/H1N1 samples from the 2005-2006, 2006-2007, and 2007-2008 influenza seasons. Data from the new assay closely matched available drug resistance genotype data previously determined by dideoxy sequencing. The H274Y mutation was only found in samples from the 2007-2008 season.

0 Followers
 · 
69 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Not Available
    Antennas and Propagation Society International Symposium, 1963; 08/1963
  • [Show abstract] [Hide abstract]
    ABSTRACT: Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.
    Journal of virological methods 02/2011; 173(2):259-65. DOI:10.1016/j.jviromet.2011.02.014 · 1.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Most clinical samples collected for diagnostic influenza testing and monitoring require refrigerated or frozen storage or shipment, which imparts logistic and cost burdens. The ability to store and ship dried clinical specimens under ambient conditions for influenza testing would significantly reduce costs and protect samples from improper storage or equipment failure, especially in remote or resource-limited areas. To evaluate the collection and storage of dried clinical samples on a transport matrix (ViveST™, ST) for influenza RNA testing by real-time reverse-transcription PCR (RT-PCR). Viral transport medium from swab or sputum samples was applied to ST, dried, and stored under ambient conditions from 2 days to 6 months. Additional aliquots of samples were frozen. Testing of frozen and ST-stored samples was performed using the WHO/CDC real-time influenza A (H1N1) RT-PCR protocol and compared to the Luminex xTAG RVP assay. ST-stored samples yielded slightly higher threshold cycle values (median 2·54 cycles) compared to frozen samples tested in parallel. This difference was consistent regardless of viral input. There was no significant difference in signal recovery between samples stored for 1 week versus samples stored for 3 weeks, or from three samples stored for 6 months. Qualitatively, clinical specimens stored on ST were 100% concordant (36/36) with frozen samples for detecting the presence of influenza A RNA. ST-processed dried specimens produced similar rates of seasonal or novel 2009 HIN1 influenza RNA detection compared to conventional sample processing and thus presents a viable alternative to refrigerated or frozen samples.
    Influenza and Other Respiratory Viruses 04/2011; 5(6):413-7. DOI:10.1111/j.1750-2659.2011.00253.x · 1.90 Impact Factor
Show more

Preview

Download
0 Downloads
Available from