Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus

Posthuma, Clara C.; Beek, Martha T. van der; Brouwer, Caroline S. de; Heiden, Pim L.J. van der; Marijt, Erik W.A.; Spaan, Willy J. M.; Claas, Eric C. J.; Nederstigt, Christa; Vossen, Ann C.T.M.; Snijder, Eric J.; Kroes, Aloys C.M.

doi:10.1016/j.jcv.2011.01.015

Cited by 11 publications

(8 citation statements)

References 40 publications

(39 reference statements)

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“…The technique is currently too elaborate and expensive for routine use, and will require much future research to validate. There is continued interest in other sequencing technologies with potential for general use in clinical diagnostic genotyping [18]. …”

Section: Laboratory Assays For Drug Resistancementioning

confidence: 99%

The biology of cytomegalovirus drug resistance

Hakki

Chou

2011

Current Opinion in Infectious Diseases

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Purpose of review To assess recently published data on cytomegalovirus (CMV) antiviral drug resistance Recent findings Resistance is typically encountered after prolonged ganciclovir treatment for post-transplant primary CMV infection, and is diagnosed by the detection of characteristic mutations in the viral UL97 kinase and UL54 DNA polymerase genes in clinical specimens. One of seven canonical UL97 mutations is detected in most cases of ganciclovir resistance, but many viral sequence variants of unknown relevance are being reported after drug exposure in vitro and in vivo. Rapid technical advances in recombinant phenotyping have shown that many of these variants confer no detectable drug resistance, while some unusual resistance mutations are newly confirmed. All currently marketed CMV antiviral drugs, including foscarnet and cidofovir, target the viral DNA polymerase, and cross-resistance may result from some UL54 mutations. To decrease cross-resistance and toxicity, there is an ongoing effort to develop anti-CMV drugs with different resistance pathways and alternative targets, such as the UL97 kinase or UL56-UL89 terminase enzymes. Summary An increasing volume of information correlating CMV genotypes and drug susceptibility phenotypes is becoming available. This will improve the interpretation of sequence-based assays currently used for clinical diagnosis, and guide the development of new antiviral drugs.

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Section: Laboratory Assays For Drug Resistancementioning

confidence: 99%

The biology of cytomegalovirus drug resistance

Hakki

Chou

2011

Current Opinion in Infectious Diseases

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“…The pH1N1 primers were designed to cover potential markers of virulence (PB2 gene positions 271, 590, 591, 627, 701; PB1-F2 gene position 66; NS1 gene positions 227–230), protein segment truncation (PB1-F2 gene positions 12, 58, 88; NS1 gene position 220) and antiviral resistance (NA gene positions 116, 117, 119, 136, 150, 151, 199, 223, 275 and 295) in the viral genome (Table 2) [11]–[14], [25], [26]. PCR amplification of the targets was performed using 2 μl of RT product, with the tagged primers in 10 μl volume in 384 well plates as previously described [19]. The sample was processed by shrimp alkaline phosphatase treatment, in vitro transcription, and C- or U-specific RNaseA cleavage, according to the manufacturer’s instructions and using a MassARRAY Liquid Handler (Matrix+Fusio™ Chip Module, Sequenom).…”

Section: Methodsmentioning

confidence: 99%

“…Wide-scale molecular surveillance is warranted but conventional sequencing methods are laborious and inefficient. Mass spectrometry-based comparative sequence analysis (MSCSA) enables rapid multigenomic sequence analysis with automated data analysis and could improve the availability of relevant pH1N1 genomic sequences [19], [20]. Rapid identification of relevant pH1N1 mutations in the community or in clinical settings may guide early prevention and intervention strategies.…”

Section: Introductionmentioning

confidence: 99%

“…Rapid identification of relevant pH1N1 mutations in the community or in clinical settings may guide early prevention and intervention strategies. MSCSA was previously successfully used for the analysis of human cytomegalovirus sequence polymorphisms in UL97 gene and for detection of genetic mutations associated with antiviral resistance in clinical specimens [19].…”

Section: Introductionmentioning

confidence: 99%

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Mass Spectrometry-Based Comparative Sequence Analysis for the Genetic Monitoring of Influenza A(H1N1)pdm09 Virus

et al. 2014

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The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.

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“…The implication of the increased sensitivity in clinical settings is still unclear. In addition, the application of mass spectrometry-based comparative sequencing was successfully used to detect ganciclovir-resistant HCMV [11]. In conclusion, more studies are required on the dynamic of virus populations under drug pressure.…”

Section: New Aspects For Early Detection Of Resistant Cytomegalovirusmentioning

confidence: 99%