An HIV-1 integrase genotype assay for the detection of drug resistance mutations
Anna C. Hearps A D , Vicki Greengrass A , Jennifer Hoy B C and Suzanne M. Crowe A B CA Clinical Research Laboratory, Centre for Virology, Burnet Institute for Medical Research and Public Health, Melbourne, Vic. 3004, Australia.
B Department of Medicine, Monash University, Melbourne, Vic. 3004, Australia.
C Infectious Diseases Unit, The Alfred Hospital, Melbourne, Vic. 3004, Australia.
D Corresponding author. Email: annah@burnet.edu.au
Sexual Health 6(4) 305-309 https://doi.org/10.1071/SH09041
Submitted: 21 April 2009 Accepted: 11 August 2009 Published: 13 November 2009
Abstract
Background: The integrase inhibitors (e.g. Raltegravir) are a new class of antiretroviral drugs that have recently become available for the treatment of patients with multi-drug resistant HIV-1 within Australia. The emergence of mutations that confer resistance to the integrase inhibitors has been observed in vivo; however, no commercial genotyping assay is currently available to screen for resistance to these drugs. Methods: The HIV-1 integrase gene was amplified from plasma-derived HIV-1 viral RNA via reverse transcription-polymerase chain reaction and genotype determined via population DNA sequencing. Drug resistance mutations and polymorphisms were detected using the Stanford University online HIV database. Assay sensitivity and reproducibility were determined using clinical and laboratory-derived samples. Results: Our in-house assay was capable of genotyping the integrase gene from all samples tested (n = 30) of HIV-1 subtypes B, C, D, F, CFR01_AE and CRF02_AG and can amplify the integrase region from plasma samples containing as few as 50 HIV RNA copies/mL. The assay is highly reproducible (average nucleotide concordance = 99.6%, n = 4) and is capable of detecting resistance-associated mutations. Conclusions:This assay is suitable for routine drug resistance screening of plasma samples from HIV-infected patients receiving integrase inhibitor antiretroviral drugs and also serves as a useful research tool.
Additional keywords: HIV drug resistance, HIV integrase genotyping, integrase polymorphism.
Acknowledgements
The present work was supported in part by a research grant from the Investigator Initiated Studies Program of Merck. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck & Co., Inc. The authors wish to thank Professor Chris Birch at the Victorian Infectious Diseases Reference Laboratory for supplying plasma samples for validation purposes and are grateful to the Victorian HIV Blood and Tissue Storage Bank for supplying samples for assay development.
[1] Steigbigel RT, Cooper DA, Kumar PN, Eron JE, Schechter M, Markowitz M, et al. Raltegravir with optimized background therapy for resistant HIV-1 infection. N Engl J Med 2008; 359 339–54.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[2] Marr P, Walmsley S. Reassessment of enfuvirtide’s role in the management of HIV-1 infection. Expert Opin Pharmacother 2008; 9 2349–62.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[3] Charpentier C, Karmochkine M, Laureillard D, Tisserand P, Belec L, Weiss L, et al. Drug resistance profiles for the HIV integrase gene in patients failing raltegravir salvage therapy. HIV Med 2008; 9 765–70.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[4] Malet I, Delelis O, Valantin MA, Montes B, Soulie C, Wirden M, et al. Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother 2008; 52 1351–8.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[5] Marinello J, Marchand C, Mott BT, Bain A, Thomas CJ, Pommier Y. Comparison of raltegravir and elvitegravir on HIV-1 integrase catalytic reactions and on a series of drug-resistant integrase mutants. Biochemistry 2008; 47 9345–54.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[6] Myers RE, Pillay D. Analysis of natural sequence variation and covariation in human immunodeficiency virus type 1 integrase. J Virol 2008; 82 9228–35.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[7] Cooper DA, Steigbigel RT, Gatell JM, Rockstroh JK, Katlama C, Yeni P, et al. Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection. N Engl J Med 2008; 359 355–65.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[8] Malet I, Delelis O, Soulie C, Wirden M, Tchertanov L, Mottaz P, et al. Quasispecies variant dynamics during emergence of resistance to raltegravir in HIV-1-infected patients. J Antimicrob Chemother 2009; 63 795–804.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[9] Lataillade M, Chiarella J, Kozal MJ. Natural polymorphism of the HIV-1 integrase gene and mutations associated with integrase inhibitor resistance. Antivir Ther 2007; 12 563–70.
| PubMed |
[10]
[11]
[12] Cozzi-Lepri A, Phillips AN, Ruiz L, Clotet B, Loveday C, Kjaer J, et al. Evolution of drug resistance in HIV-infected patients remaining on a virologically failing combination antiretroviral therapy regimen. AIDS 2007; 21 721–32.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
[13] Klibanov OM. Elvitegravir, an oral HIV integrase inhibitor, for the potential treatment of HIV infection. Curr Opin Investig Drugs 2009; 10 190–200.
| PubMed |
