Medical Microbiology and Immunology

, Volume 200, Issue 2, pp 109–113 | Cite as

Pyrosequencing allows the detection of emergent ganciclovir resistance mutations after HCMV infection

  • Susanne E. Kampmann
  • Birgit Schindele
  • Luise Apelt
  • Christoph Bührer
  • Lars Garten
  • Katharina Weizsaecker
  • Detlev H. Krüger
  • Bernhard Ehlers
  • Jörg HofmannEmail author
Original Investigation


Prenatally acquired human cytomegalovirus (HCMV) infection is the most frequent viral infection of newborns in developed countries. Virostatic therapy is accompanied by side effects and stepwise emergence of resistant virus variants. Different genotypic approaches show limited sensitivity in detecting on-growing minor resistant virus populations. Here, we demonstrate the superiority of pyrosequencing for the monitoring of mutant emergence. In a preterm baby born after 28 weeks of gestation and suffering from disseminated congenital HCMV infection, long-term control could not be achieved under ganciclovir/valganciclovir therapy and the infant died on the 113th day of life. Resistance-associated mutations in the HCMV UL97 gene were not detected by conventional DNA sequencing but postmortem pyrosequencing. Four different CMV variants carrying resistance-associated mutations each representing 11–17% of the total CMV population were found.


HCMV infection Resistance-associated mutations Pyrosequencing 



The authors wish to thank Dr. M. Raftery (Institute of Medical Virology Charité, Berlin) for critical reading of the manuscript. Expert technical assistance by Mrs. C. Stephan, S. Liebmann, and C. Walter is gratefully acknowledged.

Conflict of interest

The authors have declared that no conflict of interests exist.


  1. 1.
    Schleiss MR, McVoy MA (2004) Overview of congenitally and perinatally acquired cytomegalovirus infections: recent advances in antiviral therapy. Expert Rev Anti Infect Ther 2:389–403PubMedCrossRefGoogle Scholar
  2. 2.
    Boppana SB, Fowler KB, Britt WJ, Stagno S, Pass RF (1999) Symptomatic congenital cytomegalovirus infection in infants born to mothers with preexisting immunity to cytomegalovirus. Pediatrics 104:55–60PubMedCrossRefGoogle Scholar
  3. 3.
    Kimberlin DW, Lin CY, Sanchez PJ et al (2003) Effect of ganciclovir therapy on hearing in symptomatic congenital cytomegalovirus disease involving the central nervous system: a randomized, controlled trial. J Pediatr 143:16–25PubMedCrossRefGoogle Scholar
  4. 4.
    Schuurman R, Demeter L, Reichelderfer P, Tijnagel J, de Groot T, Boucher C (1999) Worldwide evaluation of DNA sequencing approaches for identification of drug resistance mutations in the human immunodeficiency virus type 1 reverse transcriptase. J Clin Microbiol 37:2291–2296PubMedGoogle Scholar
  5. 5.
    Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11PubMedCrossRefGoogle Scholar
  6. 6.
    Fox S, Filichkin S, Mockler TC (2009) Applications of ultra-high-throughput sequencing. Methods Mol Biol 553:79–108PubMedCrossRefGoogle Scholar
  7. 7.
    Mori T, Okamoto S, Matsuoka S et al (2000) Risk-adapted pre-emptive therapy for cytomegalovirus disease in patients undergoing allogeneic bone marrow transplantation. Bone Marrow Transplant 25:765–769PubMedCrossRefGoogle Scholar
  8. 8.
    Prix L, Hamprecht K, Holzhuter B, Handgretinger R, Klingebiel T, Jahn G (1999) Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child. J Infect Dis 180:491–495PubMedCrossRefGoogle Scholar
  9. 9.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  10. 10.
    Schindele B, Apelt L, Hofmann J et al (2010) Improved detection of mutated HCMV UL97 by pyrosequencing. Antimicrob Agents Chemother (in press)Google Scholar
  11. 11.
    Galli L, Novelli A, Chiappini E et al (2007) Valganciclovir for congenital CMV infection: a pilot study on plasma concentration in newborns and infants. Pediatr Infect Dis J 26:451–453PubMedCrossRefGoogle Scholar
  12. 12.
    Gilbert C, Boivin G (2005) Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother 49:873–883PubMedCrossRefGoogle Scholar
  13. 13.
    Bousfiha A, Picard C, Boisson-Dupuis S et al (2010) Primary immunodeficiencies of protective immunity to primary infections. Clin Immunol 135:204–209PubMedCrossRefGoogle Scholar
  14. 14.
    Casrouge A, Zhang SY, Eidenschenk C et al (2006) Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 314:308–312PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang SY, Jouanguy E, Ugolini S et al (2007) TLR3 deficiency in patients with herpes simplex encephalitis. Science 317:1522–1527PubMedCrossRefGoogle Scholar
  16. 16.
    Bassiri H, Janice Yeo WC, Rothman J et al (2008) X-linked lymphoproliferative disease (XLP): a model of impaired anti-viral, anti-tumor and humoral immune responses. Immunol Res 42:145–159PubMedCrossRefGoogle Scholar
  17. 17.
    Castor J, Cook L, Corey L et al (2007) Rapid detection directly from patient serum samples of human cytomegalovirus UL97 mutations conferring ganciclovir resistance. J Clin Microbiol 45:2681–2683PubMedCrossRefGoogle Scholar
  18. 18.
    Chou S (2008) Cytomegalovirus UL97 mutations in the era of ganciclovir and maribavir. Rev Med Virol 18:233–246PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Susanne E. Kampmann
    • 1
    • 5
  • Birgit Schindele
    • 2
  • Luise Apelt
    • 2
  • Christoph Bührer
    • 1
  • Lars Garten
    • 1
  • Katharina Weizsaecker
    • 4
  • Detlev H. Krüger
    • 3
  • Bernhard Ehlers
    • 2
  • Jörg Hofmann
    • 3
    Email author
  1. 1.Department of NeonatologyCharité UniversitätsmedizinBerlinGermany
  2. 2.Division of Viral InfectionsRobert Koch-InstitutBerlinGermany
  3. 3.Institute of Medical Virology, Helmut-Ruska-Haus, Charité UniversitätsmedizinBerlinGermany
  4. 4.Department of ObstetricsCharité University Medical CenterBerlinGermany
  5. 5.Department of PediatricsPhilipps UniversityMarburgGermany

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