Study of hepatitis B virus quasispecies by ultra-deep pyrosequencing: Resolving the nitty-gritty

Authors

  • Francisco-Rodriguez Frias M.D.,

    1. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
    2. Biochemistry Department, Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
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  • David Tabernero M.S.,

    1. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
    2. Biochemistry Department, Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
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  • Rafael Esteban M.D.,

    1. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
    2. Liver Unit, Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
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  • Maria Buti M.D.

    1. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
    2. Liver Unit, Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
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Errata

This article is corrected by:

  1. Errata: Correction: Study of hepatitis B virus quasispecies by ultra-deep pyrosequencing: resolving the Nitty-Gritty Volume 60, Issue 3, 1117, Article first published online: 25 August 2014

  • Potential conflict of interest: Nothing to report.

To the Editor:

Rodriguez et al.[1] described the dynamics of hepatitis B virus (HBV) resistance to adefovir by ultra-deep pyrosequencing (UDPS), a validated technique to study HBV quasispecies.[2] Their article reports relevant results, which, however, require further examination.

HBV polymerase variants resistant to nucleos(t)ide (NUC) treatment (NUCsRV) were detected in 83% of NUC-naïve cases, in keeping with previous reports, with prevalence restricted by sensitivity: 83%-100% at 0.03% cutoff, 12%-20% at 1% cutoff (Table 1). The authors describe an interesting computational method assuming exponential outgrowth of selected variants. Nonetheless, the relatively low coverage of each sample, at best 4,853 sequences, is questionable with respect to the lowest cutoff reported (0.03%) because variants represented by a single sequence (e.g., patient AH, with viral load 2.1 log IU/mL and 0.34% of rtT184S/A/I/L) would be considered correct, a singular notion that is not backed by previous experience (Table 1). Thus, UDPS standardization (e.g., minimal viral load and sequence coverage for standardized sensitivity, computational algorithms) must still be resolved.

Table 1. Results for Baseline Presence of NUC-Resistant Variants in All Naive Cases From Several Ultra-Deep Pyrosequencing Studies
  SamplertV173LrtL180MrtA181T/VrtT184S/A/I/LrtS202GrtM204V/I 
  1. < Percentage below the cutoff.

  2. a

    Although the empirical distribution of mismatch errors in the clone yielded an average of 0.007%, but in four positions, errors were higher than 0.02% and lower than 0.03%,which was used as cutoff. The error rates for each specific analyzed position correspond to that observed in the DNA clone.

  3. ND, variants not analyzed.

  4. b

    Possibly not naïve cases (patients C and AA).

Solmone M et al. J Virol 2009; 83:1718-1726 Reads: 9329 11<<<<<1.5One of five (20%) naïve cases with resistant variants, rates ≥1%
Error rate 0.120.120.120.120.120.12
Cutoff 111111
Margeridon-Thermet S et al. J Infect Dis 2009; 199:1275-1285 Average reads: 2900 A7<<<<<1.3Two of 17 (12%) naïve cases with resistant variants, rates ≥1%
 E6<<1<<1
Error rate 0.270.270.270.270.270.27
Cutoff 1 1111
Rodriguez-Frias F et al. PLoS One 2012; 7:e37874 Average reads: 35395 1<<0.080.110.060.13All four naïve cases (100%) with resistant variants ≥0.03%
 2<<0.180.14<0.44
 3<<0.100.070.13
 40.05<0.130.03<0.08
Error rate a 000.0190.0080.0020.008
Cutoff 0.030.030.030.030.030.03
Homs M et al. Nucleic Acids Res 2011; 39:8457-8471 Average reads: 24553 1NDNDNDNDND0.2All four (100%) naïve cases with resistant variants, rates ≥0.03%
 2NDNDNDNDND0.28
 3NDNDNDNDND0.07
 4NDNDNDNDND0.55
Error rate NDNDNDNDND0.015
Cutoff NDNDNDNDND0.03
Rodriguez C et al. Hepatology 2013; Mar 15. doi: 10.1002/hep.26383. Average reads: 4010Adefovir treatment failure1<<0.47<<0.31All seven (100%) cases with resistant variants, rates ≥0.03% to 0.21%†
2<<0.18<<<
3<<0.22<<0.32
4<<<0.17<<
5<<0.33<<<
6<<<<<<
7<<0.33<<<
Adefovir treatment successA<<0.47<<<All five naïve (100%) cases with resistant variants, rates ≥0.03% to 0.21%†
B<<<0.34<<
C†<6.3<<<9.57
D<<<<0.31<
E<<0.39<<0.37
Treatment-naïve patientsAA†<<11.290.17<<Seven of 11 (64%) naïve cases with resistant variants, rates ≥0.03% to 0.21%†
AB<<<0.35<0.32
AC<<<0.29<0.34
AD<<<<<0.27
AF<<0.18<<<
AH<<<0.34<1.79
AJ<<0.30.3<1.02
Error rate 0.120.120.120.120.120.12
Cutoff 0.050.030.080.150.180.21

The authors state that some previous UDPS studies assessing preexisting NUCsRV, including ours,[3] have methodological flaws: lack of sensitivity, no error rate consideration, and/or no linkage studies. This is surprising, because we reported the lowest cutoff to date (0.03%), established by clonal sequence processing and Poisson filtering.[3] Rodriguez et al. report different cutoffs for each variant, 0.03% being the lowest, whereas in our study 0.03% was used for all positions (0.007% average clonal error). So sensitivity was not lacking. Furthermore, we did perform a linkage study. The analysis demonstrated HBV quasispecies dynamics in a sequentially NUC-treated patient, with emergence of complex combined variants, particularly after entecavir.

Lastly, although it is a secondary issue, questions also arise about patient classification in their study. The 9.57% of rtM204V/I at baseline in “naïve” patient C suggests prior lamivudine treatment (as the authors indicate). Similarly, the 11% rtA181T/V in “naïve” patient AA, 100 times greater than previously UDPS-detected at baseline (Table 1), also suggests prior NUC therapy. As the study investigates pretreatment HBV variants, inclusion of these cases, detectable by routine methods, raises the concern that some variant populations may have been overestimated.

In conclusion, UDPS is consolidated for HBV quasispecies research, as supported by Rodriguez et al. However, certain factors, such as those mentioned here, should be kept in mind to avoid misinterpretation of the state-of-the-art in UDPS studies.

  • Francisco-Rodriguez Frias, M.D.1,2

  • David Tabernero, M.S.1,2

  • Rafael Esteban, M.D.1,3

  • Maria Buti, M.D.1,3

  • 1Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Instituto de Salud Carlos IIIMadrid, Spain

  • 2Biochemistry Department Hospital Universitari Vall d'Hebron (HUVH)Vall d'Hebron Institut de Recerca (VHIR)Universitat Autònoma de Barcelona (UAB)Barcelona, Spain

  • 3Liver Unit Hospital Universitari Vall d'Hebron (HUVH)Vall d'Hebron Institut de Recerca (VHIR)Universitat Autònoma de Barcelona (UAB)Barcelona, Spain

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