Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

S.C. Aitken; A. Kliphuis; C.L. Wallis; M.L.J.N Chu; Q. Fillekes; R. Barth; W. Stevens; T.F. Rinke de Wit; R. Schuurman

doi:10.1016/j.jcv.2012.01.010

Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

S.C. Aitken, A. Kliphuis, C.L. Wallis, M.L.J.N Chu, Q. Fillekes, R. Barth, W. Stevens, T.F. Rinke de Wit, R. Schuurman

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

BACKGROUND: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes.

OBJECTIVES: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping.

STUDY DESIGN: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs).

RESULTS: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful.

CONCLUSIONS: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).

Original language	English
Pages (from-to)	21-25
Number of pages	5
Journal	Journal of Clinical Virology
Volume	54
Issue number	1
DOIs	https://doi.org/10.1016/j.jcv.2012.01.010
Publication status	Published - 2012

Keywords

Adolescent
Adult
Africa
Child
Child, Preschool
DNA Primers
Drug Resistance, Viral
Female
HIV Infections
HIV Protease
HIV Reverse Transcriptase
HIV-1
Humans
Male
Microbial Sensitivity Tests
Polymerase Chain Reaction
Sensitivity and Specificity
Young Adult

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10.1016/j.jcv.2012.01.010

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Aitken, S. C., Kliphuis, A., Wallis, C. L., Chu, M. L. J. N., Fillekes, Q., Barth, R., Stevens, W., Rinke de Wit, T. F., & Schuurman, R. (2012). Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes. Journal of Clinical Virology, 54(1), 21-25. https://doi.org/10.1016/j.jcv.2012.01.010

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title = "Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes",

abstract = "BACKGROUND: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes.OBJECTIVES: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping.STUDY DESIGN: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs).RESULTS: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful.CONCLUSIONS: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).",

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author = "S.C. Aitken and A. Kliphuis and C.L. Wallis and M.L.J.N Chu and Q. Fillekes and R. Barth and W. Stevens and {Rinke de Wit}, T.F. and R. Schuurman",

year = "2012",

doi = "10.1016/j.jcv.2012.01.010",

language = "English",

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TY - JOUR

T1 - Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

AU - Aitken, S.C.

AU - Kliphuis, A.

AU - Wallis, C.L.

AU - Chu, M.L.J.N

AU - Fillekes, Q.

AU - Barth, R.

AU - Stevens, W.

AU - Rinke de Wit, T.F.

AU - Schuurman, R.

PY - 2012

Y1 - 2012

N2 - BACKGROUND: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes.OBJECTIVES: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping.STUDY DESIGN: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs).RESULTS: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful.CONCLUSIONS: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).

AB - BACKGROUND: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes.OBJECTIVES: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping.STUDY DESIGN: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs).RESULTS: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful.CONCLUSIONS: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).

KW - Adolescent

KW - Adult

KW - Africa

KW - Child

KW - Child, Preschool

KW - DNA Primers

KW - Drug Resistance, Viral

KW - Female

KW - HIV Infections

KW - HIV Protease

KW - HIV Reverse Transcriptase

KW - HIV-1

KW - Humans

KW - Male

KW - Microbial Sensitivity Tests

KW - Polymerase Chain Reaction

KW - Sensitivity and Specificity

KW - Young Adult

U2 - 10.1016/j.jcv.2012.01.010

DO - 10.1016/j.jcv.2012.01.010

M3 - Article

C2 - 22306272

SN - 1386-6532

VL - 54

SP - 21

EP - 25

JO - Journal of Clinical Virology

JF - Journal of Clinical Virology

IS - 1

ER -

Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

Abstract

Keywords

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