Genotypic Resistance in HIV-infected Patients Failing a d4t/3tc/nvp Regimen

Original Article Genotypic Resistance in HIV-infected Patients Failing a d4t/3tc/nvp Regimen Somnuek Sungkanuparph, M.D.* Weerawat Manosuthi, M.D.* Sasisopin Kiertiburanakul, M.D.* Wasun chantratita, Ph.D.** ABSTRACT The objectives of this study were to determine the genotypic patterns of resistance in antiretroviralnaïve patients failing initial therapy with a stavudine (d4t)/lamivudine (3TC)/nevirapine (NVP) regimen. There were 16 patients (10 females and 6 males) with the mean age of 30.3+6.5 years. Mean duration from the initiation of d4t/3tc/nvp regimen to virological failure was 20.0+13.5 months. Mean CD4 cell counts and median plasma HIV RNA at the date of virological failure were 367.6+340.1 cells/mm 3 and 5,650 copies/ml (range, 949-49,086), respectively. Twelve of 16 (75%) patients had genotypic resistance to both nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI). NRTI key mutations were M184V (87.5%), D67N (18.8%), K70R (12.5%), T215Y (12.5%), and K219Q (12.5%). No patient developed mutations associated with didanosine (ddi) or tenofovir disoproxil fumurate (TDF) resistance (K65R or L74V). NNRTI key mutations were K103N (50%), Y181C (18.8%), G190A/S (18.8%) and V108I (12.5%). Selection of NVP and 3TC resistance often with other thymidine analogue mutations (TAMs) was frequently associated with virological failure in our patients. Genotypic resistance testing is helpful for selection of the next regimen in patients failing d4t/3tc/nvp regimen. The prospective genotypic resistance study with a larger number of patients is required to confirm our results. (J Infect Dis Antimicrob Agents 2004;21:61-7.) INTRODUCTION However, long-term suppression of HIV is not easy to The use of highly active antiretroviral therapy achieve because of the adverse effects, complexity of (HAART) has dramatically changed the course of drug regimens and drug costs, which adversely impact human immunodeficiency virus (HIV) disease, with adherence, leading to selection of resistant mutants and a substantial reduction in morbidity and mortality. 1 *Division of Infectious Diseases, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. *Molecular Virology Unit, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. Received for publication: February 24, 2004. Reprint request: Somnuek Sungkanuparph, M.D., Division of Infectious Diseases, Department of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. Keywords: HIV, genotypic resistance, stavudine, lamivudine, nevirapine, GPO-VIR 61

62 J INFECT DIS ANTIMICROB May-Aug. 2004 treatment failure. In April 2002, the Thai Government Pharmaceutical Organization (GPO) produced a generic combined pill of three anti-hiv drugs, stavudine (d4t), lamivudine (3TC), and nevirapine (NVP), called GPO- VIR. The cost of GPO-VIR is lower than the original pills, and is much more affordable for HIV-infected patients in Thailand. In 2004, 60,000 HIV-infected people are expected to use this combined pill. 2 Thus, a large number of patients who will fail from this single regimen in the near future is anticipated. HIV resistance to antiretroviral agents is a major contributory cause of treatment failure. The dynamics of HIV replication, together with patient-, physician-, and drug-related factors, lead to emergence of HIVresistant strains in some patients. GPO-VIR, an NVP-containing regimen, is simple and well tolerated but have a low genetic barrier that may facilitate the development of high-level resistance in only one step of viral mutation. 3 HIV genotypic resistance testing, which detects resistance mutations in the reverse transcriptase and protease genes by comparing the gene sequences of resistant virus to those of a wildtype strain that has been previously described. The efficacy of each antiretroviral class and each individual antiretroviral drug is threatened by specific mutations and resistance mechanisms. 4 Genotypic resistance testing is most useful in patients failing an initial regimen and in identifying the presence of resistance to drugs in the currently failing regimen. 5-8 However, the cost of the test limits the usefulness of resistance testing in clinical practice in Thailand. Our study aimed to demonstrate the genotypic resistance patterns in patients who failed from the specific regimen of d4t/3tc/nvp, and suggest the options of salvage therapy in patients who failed GPO-VIR and cannot afford genotypic resistance testing. PATIENTS AND METHODS Patients From January to December 2003, HIVinfected patients who had virological failure from an initial regimen of d4t/3tc/nvp and were tested for HIV genotypic resistance in Ramathibodi Hospital were included into the study. According to the US and British treatment guidelines, the definition of virological failure includes no achieving undetectable HIV RNA (<400 copies/ml) at 24 weeks of treatment or rebound HIV RNA during treatment. 9,10 The patterns of drugresistant viral genotypes, mutations critical to the HIV- 1 protease and reverse transcriptase sequences were reviewed from the results of genotypic resistance tests. Individuals who had been on drug holidays for longer than 2 weeks before the time of genotypic resistance tests were excluded. Genotypic Resistance Testing HIV RNA was extracted from plasma samples, using the QIAamp viral extraction kit (Qiagen, Inc., Chatsworth, CA, USA). The TRUGENE HIV-1 Genotyping Assay 11,12 was used in conjunction with the Open Gene automated DNA sequencing system (Visible Genetics Inc., Toronto, Canada) to sequence the protease and reverse transcriptase (RT) regions of the HIV-1 cdna. Testing involved simultaneous clip sequencing of protease and codons 35-244 of the RT from the amplified cdna in both the 3 and 5 directions. Sequences were aligned and compared with a lymphoadenopathy-associated virus type 1 (HIV-B-LAV1) consensus sequence using Visible Genetics Gene Librarian software. We focused on mutation at positions in the polymerase gene known to be associated with the commonly used drugs against HIV-1. Interpretation of the genotype in terms of drug resistance was based on an algorithm established by the Stanford HIV RT and Protease Sequence Database. 13 RESULTS There were 16 patients with 10 (62.5%) females and 6 males. The mean age was 30.3+6.5 years. Ten patients had received GPO-VIR, the others had separate pills of d4t, 3TC and NVP. Mean duration from the initiation of d4t/3tc/nvp regimen to virological failure was 20.0+13.5 months. Mean CD4 cell counts and median plasma HIV RNA at the date of virological failure were 367.6+340.1 cells/mm 3 and

5,650 copies/ml (range, 929-49086), respectively. All patients had genotypic resistance to nucleoside reverse transcriptase inhibitor (NRTI) or non-nucleoside reverse transcriptase inhibitor (NNRTI). Twelve of 16 (75%) patients had genotypic resistance to both NRTI and NNRTI. NRTI key mutations were recognized in 14 (87.5%) patients. These included M184V (87.5%), D67N (18.8%), K70R (12.5%), T215Y (12.5%), and K219Q (12.5%) (Figure 1). No patients developed mutations associated with didanosine (ddi) or tenofovir disoproxil fumarate (TDF) resistance (K65R or L74V). NNRTI key mutations were recognized in 14 (87.5%) patients. These included K103N (50%), Y181C (18.8%), G190A/S (18.8%) and V108I (12.5%) (Figure 2). Genotypic resistance to protease inhibitor (PI) was not detected. DISCUSSION HIV drug resistance results mainly from the interplay of HIV replication and antiretroviral drug selection pressure. When antiretroviral therapy does not completely suppress viral replication, the replicating HIV population has the opportunity to develop new mutations. The duration of virus suppression depends Figure 1. Genotypic resistance to NRTI key muta- Figure 2. Genotypic resistance to NNRTI key mutations. 63

64 J INFECT DIS ANTIMICROB May-Aug. 2004 on the number of viral mutations required to overcome the antiviral activity of the regimen (genetic barrier) and the rate of replication (fitness) of drug-resistant virus. A regimen with high genetic barrier, e.g. PI-based regimen, would require the virus population to acquire multiple mutations before development of virological failure. In contrast, NNRTI-based regimens have a low genetic barrier and allow the development of highlevel resistance in one step of mutation. 3 A regimen of d4t/3tc/nvp, currently the most commonly prescribed regimen in Thailand, contains 2 lowgenetic-barrier drugs, NVP and 3TC. HIV acquires high resistance for these agents with only a single mutation. 14 Success of treatment from this regimen requires a strict adherence. 15,16 Our study showed that 75 percent of the patients failing a d4t/3tc/nvp regimen had key mutations to both NVP and 3TC. Twelve of 16 (87.5%) patients had key mutations to NVP. K103N occurred in a half of our patients. Other studies to determine the genotypic and phenotypic patterns of resistance in patients failing initial therapy with NVP-based regimens also showed a high prevalence of NVP resistance and demonstrated K103N as the most common mutation. 17-19 The K103N mutation indirectly inhibits the binding of NNRTI to the binding pocket of RT and results in a 20-fold resistance to NVP and efavirenz (EFV). 20,21 Therefore, mutations conferring resistance to one drug in this class generally confer cross-resistance to all other NNRTIs. Normally, NNRTI resistance mutations occur in 2 clusters in the RT gene at codons 100-108 and at codons 179-190. 20 The most common changes selected by NVP involve a K103N mutation and a Y181C mutation. 20,22 However, our study showed that only 19 percent of the patients had Y181C. Another low-genetic-barrier drug in the d4t/3tc/ NVP regimen is 3TC. Resistance to 3TC is conferred by a point mutation at RT codon 184, producing M184V or M184I. 23,24 In a study of 3TC monotherapy before the HAART era, the entire wild-type population of HIV in the plasma is replaced by variants carrying the M184V mutation within weeks of initiating treatment with 3TC. 25 3TC resistance is rapidly acquired if the other components in the regimen are not effective. It is not surprising that 88 percent of our patients had M184V. A previous study of genotypic resistance in 83 HIV-1 infected Thai patients who had been treated with any antiretroviral drug also showed that M184V/I was the most common mutations. 26 Our study also showed that there were other thymidine analogue mutations (TAMs), including D67N, K70R, T215Y, and K219Q, from the failure of d4t/3tc/nvp regimen. One patient (6.3%) had 4 TAMs (data not shown). Studies from several groups clearly demonstrates that d4t can select mutations typically associated with zidovudine (AZT) resistance (eg. K70R, T215Y). 27-30 There is substantial evidence of cross-resistance between AZT and d4t. 28-31 Presence of these mutations was significantly associated with a poor response to d4t therapy. 19,31,32 Multinucleoside drug resistance mutation including the Q151M complex and insertion mutation at codon 69, were not observed in our study, because every patient in our study had failed only one regimen containing d4t/3tc/nvp for a short period of treatment. Higher experience with multiple regimens increases the risk of multinucleoside drug resistance. 33 According to the results of our study, patients who failed a d4t/3tc/nvp regimen would also have a very high rate of resistance to all NNRTI and 3TC, and low rate of resistance to other NRTIs including d4t and AZT. The salvage regimens for these patients could be boosted PI-based regimens with two reliable NRTIs, such as ddi or TDF. However, TDF is not currently available in Thailand. Thus, abacavir (ABC) may be used. ABC retained efficacy against resistant HIV with the M184V genotype alone. 34 However, 6.3 percent of our patients had 4 TAMs. The chance of ABC failure in this salvage regimen is possible. 35 In addition, 5 of 16 (31.3%) patients had D67N, K70R, T215Y or K219Q. The chance of failure when using of AZT or d4t should also be relatively high. The limitation of our study includes a retrospective fashion and low number of patients. We did not demonstrate the sequences of mutations occuring

in our patients. These results are also based on an early detection of virological failure on the basis of HIV RNA testing every 3-6 months. In most medical centers, HIV RNA is not easily performed and virological failure is usually detected late after treatment. There could be a higher rate of NRTI resistance including multinucleoside resistances. We did not determine the adherence of patients in this study due to the retrospective fashion. In conclusion, selection of NVP and 3TC resistance often associated with other TAM mutations was frequently observed in patients failing a d4t/3tc/ NVP regimen. Genotypic resistance testing is helpful for selection of the next regimen in patients failing this regimen. A well-designed prospective study to investigate the genotypic resistance pattern in patients failing initial therapy with GPO-VIR is required. ACKNOWLEDGEMENT The authors would like to thank all the attending staffs and fellows in Division of Infectious Diseases who had cared the patients in this study, and the staffs in Molecular Virology Unit, Department of Pathology, Ramathibodi Hospital. References 1. Palella FJ Jr, Delaney KM, Moorman AC, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998;338:853-60. 2. Cohen J. Thailand s do-it-yourself therapy. Science 2003;301:1662. 3. Podzamczer D, Fumero E. The role of nevirapine in the treatment of HIV-1 disease. Expert Opin Pharmacother 2001;2:2065-78. 4. Johnson VA, Brun-Vezinet F, Clotet B, et al. Drug resistance mutations in HIV-1. Top HIV Med 2003; 11:215-21. 5. Hirsch MS, Brun-Vezinet F, Clotet B, et al. Antiretroviral drug resistance testing in adults infected with human 65 immunodeficiency virus type 1: 2003 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis 2003;37:113-28. 6. Haupts S, Ledergerber B, Boni J, et al. Impact of genotypic resistance testing on selection of salvage regimen in clinical practice. Antivir Ther 2003;8:443-54. 7. Clevenbergh P, Durant J, Halfon P, et al. Persisting long-term benefit of genotype-guided treatment for HIV-infected patients failing HAART. The Viradapt Study: week 48 follow-up. Antivir Ther 2000;5:65-70. 8. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS. AIDS 2000;14:F83-93. 9. Dybul M, Fauci AS, Bartlett JG, Kaplan JE, Pau AK. Guidelines for using antiretroviral agents among HIV-infected adults and adolescents. Ann Intern Med 2002;137(5 Pt 2):381-433. 10. Pozniak A, Gazzard B, Anderson J, et al. British HIV Association (BHIVA) guidelines for the treatment of HIV-infected adults with antiretroviral therapy. HIV Med 2003;4 (Suppl. 1):1-41. 11. Kuritzkes DR, Grant RM, Feorino P, et al. Performance characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System. J Clin Microbiol 2003;41:1594-9. 12. Grant RM, Kuritzkes DR, Johnson VA, et al. Accuracy of the TRUGENE HIV-1 genotyping kit. J Clin Microbiol 2003;41:1586-93. 13. Shafer RW, Jung DR, Betts BJ, Xi Y, Gonzales MJ. Human immunodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Research 2000;28:346-8. 14. Soriano V. Sequencing antiretroviral drugs. AIDS 2001;15:547-51. 15. Podzamczer D, Ferrer E, Consiglio E, et al. A randomized clinical trial comparing nelfinavir or nevirapine associated to Zidovudine/Lamivudine in

66 J INFECT DIS ANTIMICROB May-Aug. 2004 HIV-infected naïve patients (The Combine Study). Antivir Ther 2002;7:81-90. 16. Staszewswki S, Morales-Ramirez J, Tashima KT, et al. Efavirenz plus zidovudine and lamivudine, efavirenz plus indinavir, and indinavir plus zidovudine and lamivudine in the treatment of HIV-1 infection in adults. Study 006 Team. N Engl J Med 1999;341:1865-73. 17. Re MC, Bon I, Monari P, et al. Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors. Int J Antimicrob Agents 2003;22:388-94. 18. Ferrer E, Podzamczer D, Arnedo M, et al. Genotype and phenotype at baseline and at failure in human immunodeficiency virus-infected antiretroviral-naïve patients in a randomized trial comparing zidovudine and lamivudine plus nelfinavir or nevirapine. J Infect Dis 2003;187:687-90. 19. Vidal C, Arnedo M, Garcia F, et al. Genotypic and phenotypic resistance patterns in early-stage HIV-1- infected patients failing initial therapy with stavudine, didanosine and nevirapine. Antivir Ther 2002;7:283-7. 20. Hsiou Y, Ding J, Das K, et al. The Lys103Asn mutation of HIV-1 RT: a novel mechanism of drug resistance. J Mol Biol 2001;309:437-45. 21. Spence RA, Anderson KS, Johnson KA. HIV-1 reverse transcriptase resistance to non-nucleoside inhibitors. Biochemistry 1996;35:1054-63. 22. Maga G, Amacker M, Ruel N, Hubscher U, Spadari S. Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions. J Mol Biol 1997;274:738-47. 23. Hertogs K, Bloor S, De Vroey V, et al. A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. Antimicrob Agents Chemother 2000;44:568-73. 24. Boucher CA, Cammack N, Schipper P, et al. High-level resistance to (-) enantiomeric 2 -deoxy-3 -thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 1993;37:2231-4. 25. Schuurman R, Nijhuis M, van Leeuwen R, et al. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J Infect Dis 1995;171:1411-9. 26. Chantratita W, Jenwitheesuk E, Watitpun C, et al. Prevalence of HIV-1 polymerase gene mutations in pre-treated patients in Thailand. Southeast Asian J Trop Med Public Health 2002;33:80-4. 27. Coakley EP, Gillis JM, Hammer SM. Phenotypic and genotypic resistance patterns of HIV-1 isolates derived from individuals treated with didanosine and stavudine. AIDS 2000;14:F9-15. 28. Katlama C, Valantin MA, Matheron S, et al. Efficacy and tolerability of stavudine plus lamivudine in treatment-naïve and treatment-experienced patients with HIV-1 infection. Ann Intern Med 1998;129:525-31. 29. Gilleece Y, Torti C, Mandalia S, Gazzard BG, Pillay D, Pozniak AL. The prevalence of reduced zidovudine susceptibility in zidovudine-naïve, antiretroviralexperienced HIV-1-infected patients. HIV Med 2003;4:305-10. 30. Lennerstrand J, Hertogs K, Stammers DK, Larder B. Biochemical mechanism of HIV-1 reverse transcriptase resistance to stavudine [abstract]. Antivir Ther 2000;5 (Suppl 3):14-5. 31. Calvez V, Costagliola D, Descamps D, et al. Impact of stavudine phenotype and thymidine analogues mutations on viral response to stavudine plus lamivudine in ALTIS 2 ANRS trial. Antivir Ther 2002;7:211-8. 32. Sarmati L, Nicastri E, Parisi SG, et al. Failure of stavudine-lamivudine combination therapy in antiretroviral-naïve patients with AZT-like HIV-1 resistance mutations. J Med Virol 2001;65:631-6. 33. Zaccarelli M, Perno CF, Forbici F, et al. Q151Mmediated multinucleoside resistance: prevalence, risk factors, and response to salvage therapy. Clin

Infect Dis 2004;38:433-7. 34. Whitcomb JM, Parkin NT, Chappey C, Hellmann NS, Petropoulos CJ. Broad nucleoside reversetranscriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. J Infect Dis 2003;188:992-1000. 35. Ait-Khaled M, Rakik A, Griffin P, et al. Mutations in HIV-1 reverse transcriptase during therapy with abacavir, lamivudine and zidovudine in HIV-1-infected adults with no prior antiretroviral therapy. Antivir Ther 2002;7:43-51. 67