1119. Use of RT-PCR/Electrospray Ionization Mass Spectrometry (RT-PCR/ESI-MS) to Monitor Evolution of Genotypes and Surveillance of Pandemic H1N1 Influenza in Realtime  
Session: Poster Abstract Session: Influenza and H1N1 Diagnosis, Epidemiology, and Viral Outcome
Saturday, October 22, 2011
Room: Poster Hall B1
  • IDSA Gaydos Influenza T5000 PowerPoint Poster Oct 2011.pdf (587.8 kB)
  • Background:  

    The novel H1N1 influenza pandemic (pH1N1) demonstrated the requirement for accurate, rapid tests to differentiate influenza-like illnesses (ILI), as well as a need for molecular surveillance for monitoring influenza A evolution.  RT-PCR with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) allows identification, strain-typing, and genetic analysis of influenza by using base composition characterization of amplified fragments from six target genes (PB1, NP, M1, PA, NS1, and NS2). We used this assay to identify genetic changes of pandemic H1N1 isolates over the later phases of the outbreak compared to earlier genotypes.  


    Nasopharyngeal aspirates (N =70) from patients at the Johns Hopkins Hospital that previously tested positive for influenza A pH1N1 between October - December, 2009 were tested using influenza-specific primers followed by base composition analysis of the amplicons by ESI-MS for pathogen identification.  Positive controls (pH1N1, seasonal H1N1, H3N2) from Zeptometrix, Inc. were used as positive controls for assay validation.  Results were compared to original CDC-approved RT-PCR. The genotyping data was compared to the published genotypes from earlier in the H1N1 outbreak (April-June 2009) and analyzed for genetic diversity.

    Results:  Compared to CDC RT-PCR results for typing, 69/70 (98.6%) samples were identified as pH1N1 (1 sample was insufficient quantity for analysis), and RT-PCR/ESI-MS correctly identified the pH1N1 A/California/04/2009 strain in all 69 samples. The total time to identification and genotype was 8 hours.  Late year samples exhibited greater genetic diversity (11 genotypes vs. 7 genotypes). The proportion of pandemic H1N1 samples with genotypes divergent from the reference strain increased from the uniform genotype of early samples to 61.4% in our later samples. 


    RT-PCR/ESI-MS accurately detected and typed pH1N1 influenza samples, and provided evidence that genetic mutations were occurring in the gene segments. Spectrometry data revealed an increase in genetic diversity of samples collected over time, demonstrating its potential use for molecular epidemiology of emerging influenza viruses. 

    Subject Category: V. Virology including clinical and basic studies of viral infections, including hepatitis

    Charlotte Gaydos, DrPH1, Kevin Jeng, B.S.2, Lawrence Blyn, PhD3, David Metzgar, PhD4, Justin Hardick, MS5,6, Alexandra Valsamakis, MD, PhD7, Linda Gluck, MS8, Richard Rothman, MD, PhD9 and Ranga Sampath, PhD4, (1)Johns Hopkins University, Baltimore, MD, (2)Medicine, Infectious Diseases, Johns Hopkins University, Baltimore, MD, (3)Ibis Biosciences, Inc., A Division of Abbott, Carlsbad, CA, (4)Ibis Biosciences, Inc., Carlsbad, CA, (5)Medicine ,Infectious Diseases, Johns Hopkins University, Baltimore, MD, (6)USAMRIID/GBR Fort Deterick, Frederick, MD, (7)Johns Hopkins Medical Institutions, Baltimore, MD, (8)Laboratory Medicine, Johns Hopkins Hospital, Baltimore, MD, (9)Johns Hopkins Medical Institute, Baltimore, MD


    C. Gaydos, Ibis Biosciences: Grant Investigator, Grant recipient

    K. Jeng, None

    L. Blyn, Ibis Biosciences: Employee, Salary

    D. Metzgar, Ibis Biosciences: Employee, Salary

    J. Hardick, None

    A. Valsamakis, None

    L. Gluck, None

    R. Rothman, Ibis Biosciences: Grant Investigator, Grant recipient

    R. Sampath, Ibis Biosciences: Employee, Salary

    Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.