Human immunodeficiency virus type 1 drug resistance testing: Evaluation of a new ultra-deep sequencing-based protocol and comparison with the TRUGENE HIV-1 Genotyping Kit

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Abstract

Genotypic HIV-1 drug resistance testing with standard Sanger sequencing is limited to the detection of mutations with >20% prevalence.

A new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer (Roche 454 Life Sciences, Branford, CT) was evaluated. The new technology was compared with the standard Sanger sequencing method.

For accuracy testing, genotype B samples obtained from proficiency panels were examined with ultra-deep sequencing. Reproducibility was determined by repeat GS-FLX sequencing of 21 clinical samples. Clinical performance was evaluated with 44 samples and the results were compared to the TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Sequences generated with both protocols were analyzed using the Stanford University HIV drug resistance database.

When accuracy was tested, 316 of 317 mutation codons included in the analysis of proficiency panels could be identified correctly with ultra-deep sequencing. Reproducibility testing resulted in a correlation value of R2 = 0.969. Analysis of 44 routine clinical samples with the Stanford University HIV drug resistance database revealed a total number of 269 and 171 mutations by the ultra-deep and standard Sanger sequencing, respectively. Drug resistance interpretations showed differences for 11 samples. With ultra-deep sequencing, total time to result was four times longer in comparison to standard Sanger sequencing. Manual work was increased significantly using the new protocol.

The ultra-deep sequencing protocol showed good accuracy and reproducibility. However, automation and shorter time to obtain results are essential for use in the routine diagnostic laboratory.

Highlights

► Ultra-deep sequencing may be a good alternative to Sanger sequencing in future. ► It shows good results for HIV-1 drug resistance testing. ► For use in the routine clinical lab, reduction of manual steps is necessary.

Introduction

Genotypic HIV-1 drug resistance testing has been considered an important tool for HIV-1 drug monitoring. Resistance testing is recommended at the time of HIV infection diagnosis as part of the initial comprehensive patient assessment, as well as in all patients with virological failure (Clumeck et al., 2008, Hammer et al., 2008, Little et al., 2002). Currently, commercially available HIV drug resistance genotyping kits are based on dideoxynucleotide-terminator sequencing which is unable to detect mutations with low abundance (<20% prevalence) (Erali et al., 2001, Eshleman et al., 2005). The TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY) is the only FDA-approved and IVD/CE-labeled assay for HIV-1 drug resistance testing and was reported to be useful in the routine diagnostic laboratory (Grant et al., 2003, Kuritzkes et al., 2003). Recently, a new protocol for ultra-deep sequencing of the HIV-1 protease and reverse transcriptase genes on the Genome Sequencer (GS) FLX System (Roche 454 Life Sciences, Branford, CT) based on massive parallelization of pyrosequencing was introduced (Simen et al., 2009).

The aim of this study was to evaluate the ultra-deep sequencing protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer and to compare the new technology with the TRUGENE HIV-1 Genotyping Kit (Siemens), based on standard Sanger sequencing. Accuracy and reproducibility of the ultra-deep sequencing protocol were determined. Results obtained from clinical samples were compared to those generated with the TRUGENE HIV-1 Genotyping Kit.

Section snippets

Ultra-deep sequencing

For ultra-deep sequencing, a large volume protocol was employed in order to increase the sensitivity of the technique. Samples were extracted manually with the High Pure Viral Nucleic Acid Large Volume Kit (Roche Applied Science, Penzberg, Germany). One milliliter of plasma was centrifuged for 1 h at 20,800 × g and extracted subsequently according to the package insert instructions. Nucleic acids were eluted in 75 μl elution buffer. Aliquots of 11.5 μl were used for cDNA generation.

Ultra-deep

Results

When accuracy was tested with five genotype B samples included in the QCMD ENVA 2009, 2008, and 2006 HIV Drug Resistance Typing EQA programs, 316 of 317 mutation codons included in the analysis of the proficiency panel could be identified correctly with ultra-deep sequencing (Table 1). Reproducibility testing by repeat analysis of 21 routine clinical samples showed a correlation value of R2 = 0.969 for the linear regression of the variant prevalence.

Analysis of 44 routine clinical samples with

Discussion

HIV-1 drug resistance testing has been considered an important tool for HIV-1 drug monitoring. Drug resistance assays are usually based on standard Sanger sequencing. Ultra-deep sequencing is a new tool allowing detection of mutations with prevalences <20%. The aim of this study was to evaluate a new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer and to compare results with the in vitro

Acknowledgements

The authors gratefully acknowledge Christian Lunde for helpful comments on the manuscript and Egon Marth for continuous support. Reagents required for this study were kindly provided by Roche applied science.

References (13)

  • J. Chun et al.

    The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer

    BMC Microbiol.

    (2010)
  • N. Clumeck et al.

    EACS Executive Committee, European AIDS Clinical Society (EACS) guidelines for the clinical management and treatment of HIV-infected adults

    HIV Med.

    (2008)
  • M. Erali et al.

    Human immunodeficiency virus type 1 drug resistance testing: a comparison of three sequence-based methods

    J. Clin. Microbiol.

    (2001)
  • S.H. Eshleman et al.

    Sensitivity and specificity of the ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for detection of HIV-1 drug resistance mutations by use of an ABI PRISM 3100 genetic analyzer

    J. Clin. Microbiol.

    (2005)
  • R.M. Grant et al.

    Accuracy of the TRUGENE HIV-1 Genotyping Kit

    J. Clin. Microbiol.

    (2003)
  • S.M. Hammer et al.

    International AIDS Society-USA, Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel

    JAMA

    (2008)
There are more references available in the full text version of this article.

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