High-throughput matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative approach to monitoring drug resistance of hepatitis B virus.
Long-term antiviral therapy of chronic hepatitis B virus (HBV) infection can lead to the selection of drug-resistant HBV variants and treatment failure. Moreover, these HBV strains are possibly present in treatment-naive patients. Currently available assays for the detection of HBV drug resistance can identify mutants that constitute ≥5% of the viral population. Furthermore, drug-resistant HBV variants can be detected when a viral load is >10(4) copies/ml (1,718 IU/ml). The aim of this study was to compare matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multitemperature single-strand conformation polymorphism (MSSCP) with commercially available assays for the detection of drug-resistant HBV strains. HBV DNA was extracted from 87 serum samples acquired from 45 chronic hepatitis B (CHB) patients. The 37 selected HBV variants were analyzed in 4 separate primer extension reactions on the MALDI-TOF MS. Moreover, MSSCP for identifying drug-resistant HBV YMDD variants was developed and turned out to be more sensitive than INNOLiPA HBV DR and direct sequencing. MALDI-TOF MS had the capability to detect mutant strains within a mixed viral population occurring with an allelic frequency of approximately 1% (with a specific value of ≥10(2) copies/ml, also expressed as ≥17.18 IU/ml). In our study, MSSCP detected 98% of the HBV YMDD variants among strains detected by the MALDI-TOF MS assay. The routine tests revealed results of 40% and 11%, respectively, for INNOLiPA and direct sequencing. The commonly available HBV tests are less sensitive than MALDI-TOF MS in the detection of HBV-resistant variants, including quasispecies.
Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
Magda
M
Rybicka
Piotr
P
Stalke
Marcin
M
Dreczewski
Tomasz
T
Smiatacz
Krzysztof Piotr
KP
Bielawski
D000998:Antiviral Agents / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000187:drug effects* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D044967:Serum / Q000821:virology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000998:Antiviral Agents; D004279:DNA, Viral