Clinical: Original Papers
Emergence of zidovudine and multidrug-resistance mutations in the HIV-1 reverse transcriptase gene in therapy-naive patients receiving stavudine plus didanosine combination therapy
Pellegrin, Isabelle; Izopet, Jacquesa; Reynes, Jacquesb; Denayrolles, Muriel; Montes, Brigitteb; Pellegrin, Jean-Luc; Massip, Patricea; Puel, Jacquelinea; Hervé Fleury and Michel Segondyb for the STADI Group
From the Departments of Virology and Infectious Diseases, Bordeaux University Hospital, Bordeaux, the aDepartments of Virology and Infectious Diseases, Toulouse University Hospital, Toulouse, and the bDepartments of Virology and Infectious Diseases, Montpellier University Hospital, Montpellier, France. *See Appendix.
Sponsorship: Supported by grants from Bristol-Myers Squibb (Paris-La Défense, France).
Correspondence to I. Pellegrin, Laboratoire de virologie, Hopital Pellegrin, place Amélie Raba Léon, 33076 Bordeaux Cedex, France.
Received: 9 March 1999; revised: 24 May 1999; accepted: 1 June 1999.
Objective: Assessment of genotypic changes in the reverse transcriptase gene of HIV-1 occurring in antiretroviral naive patients treated by stavudine plus didanosine combination therapy.
Methods: Sequence analysis (codons 1-230) was performed after amplification of the reverse transcriptase gene from plasma samples collected at baseline and at the end of treatment from 39 previously treatment-naive patients treated for 24-48 weeks.
Results: At baseline, mutations associated with zidovudine resistance were detected in plasma from two patients: Asp67Asn/Lys219Gln and Leu210Trp. Among the 39 subjects, 18 (46%) developed mutations: one developed the Val75Thr/Ala mutation, four (10%) developed a Gln151Met multidrug-resistance mutation (MDR), associated in one of them with the Phe77Leu and the Phe116Tyr MDR mutations and 14 (36%) developed one or more zidovudine-specific mutations (Met41Leu, Asp67Asn, Lys70Arg, Leu210Trp, Thr215Tyr/Phe). The development of a Met41Leu zidovudine-specific mutation was associated with the development of a Gln151Met mutation in one patient. Other reverse transcriptase mutations known to confer resistance to nucleoside analogues were not detected. At inclusion, there was no statistical difference in HIV-1 load between patients who developed resistance mutations and those who did not. RNA HIV-1 load decrease was higher (P=0.05) in patients who maintained a wild-type reverse transcriptase genotype (-2.22log10 copies/ml) than in patients who developed resistance mutations (-1.14log10 copies/ml).
Conclusion: Stavudine/didanosine combination therapy is associated with emergence of zidovudine-related resistance or MDR mutations in naive patients. These findings should be considered when optimizing salvage therapy for patients who have received a treatment including stavudine/didanosine combination.
A set of mutations occurring in the reverse transcriptase (RT) gene of HIV-1 confers resistance to antiretroviral nucleoside analogues acting as RT inhibitors. RT mutations conferring resistance to zidovudine (ZDV), mainly Met41Leu, Lys70Arg, and Thr215Tyr/Phe, or to lamivudine (3TC), such as Met184Val, can be detected with a high frequency in patients treated with these drugs[1,2]. In contrast, mutations which confer resistance to didanosine (ddI), such as Leu74Val, or to zalcitabine (ddC), such as Thr69Asp, are detected less frequently[3,4]. However, it has been shown that ZDV/ddI or ZDV/ddC combination therapy can select multidrug-resistance (MDR) mutations, in particular Gln151Met, which confers cross resistance to different nucleoside analogues.
Mechanisms of resistance to stavudine (d4T) are still incompletely understood. It has been reported that HIV-1 remains susceptible to d4T following prolonged d4T therapy[6,7]. Furthermore, d4T-resistant HIV-1 variants have not been characterized from treated patients, although resistance mutations have been reported following in vitro passage of HIV-1 laboratory strains in the presence of d4T. A mutation at codon 75 (Val75Thr) which confers a sevenfold increase in d4T IC50 and a reduced susceptibility to both ddI and ddC has been identified in vitro. A mutation at codon 50 (Ile50Thr) leading to a 30-fold reduction in sensitivity to d4T has also been observed in vitro. This mutation does not appear to confer cross-resistance to other nucleoside analogues. More recently, insertions at codon 69 which confer resistance to d4T have been reported.
The d4T/ddI combination is a well tolerated and relatively potent two-drug combination therapy. Nevertheless, as other dual combination therapy, this regimen is suboptimal in terms of antiviral efficacy. However, the development of RT mutations conferring drug resistance in patients treated with this regimen is still poorly documented. We therefore analysed the emergence of RT mutations in patients treated for 24-48 weeks by a d4T/ddI combination therapy.
Patients and methods
Subjects included in this study were HIV-1 seropositive antiretroviral-naive adults with 100-500×106 CD4 T cells/l. These patients were included in the STADI trial, a pilot study that evaluated the use of once daily administration of ddI (300mg in patients >60kg and 200mg in patients <60kg) in combination with twice daily administration of d4T (40mg in patients >60kg and 30mg in patients <60kg). After baseline evaluation (W0), patients were seen at week 4 (W4), week 8 (W8), and at 8 weeks intervals thereafter until week 48 (W48). At the same time, plasma and peripheral blood mononuclear cells (PBMC) were collected and stored at -80°C until use.
HIV-1 RNA quantification
Quantification of plasma HIV-1 RNA was performed at baseline, W4, W8, W24 and W48 by the Quantiplex HIV-1 RNA 2.0 assay (Chiron Diagnostics, Emeryville, California, USA) according to manufacturer‚s instructions. Plasma samples with values below the detection limit (500copies/ml) were tested by the AMPLICOR HIV-1 MONITOR assay (Roche Diagnostics, Bazel, Switzerland) with a detection limit of 20copies/ml.
Plasma RNA from W0 and W48 samples (or from the last sample available between W24 and W48) were used for sequence analysis of the RT gene (codons 1-230). HIV-1 RNA was purified from 1ml ultracentrifuged (23500 ×g) plasma using the High Pure Viral RNA Purification kit (Boehringer Mannheim, Mannheim, Germany). Plasma HIV-1 RNA was amplified by a one-step reverse transcription-PCR using the TitanTM One Tube Reverse Transcription- PCR Kit (Boehringer Mannheim) followed by a nested PCR with AmpliTaq GoldTM (Applied Biosystems, Foster City, California, USA). The primers RT19 (5′-GCA CAT AAA GCT ATA GGT ACA G-3′) and RT20 (5′-CTG CCA GTT CTA GCT CTG CTT C-3′) were used for the one-step reverse transcription-PCR, and primers MJ3 (5‚-AGT AGG ACC TAC ACC TGT CAA C-3′) and NE1(35) (5′-CCT ACT AAC TTC TGT ATG TCA TTG ACA GCT CAG CT-3′) were used for the nested PCR. The purified 776bp DNA fragment obtained was directly sequenced [primers NE1(20) (5′-ATG TCA TTG ACA GTC CAG CT-3′) and /A (5′-ATT TTC CCA TTA GTC CTA TT-3‚)] using the ABI PRISM Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Sequence products were analysed with the ABIPRISM 377 automatic sequencing system and the sequences were aligned on the HIV-1LAI RT gene using the Sequence Navigator software (Applied Biosystems). Codons changes considered as mutations were the five mutations most commonly associated to MDR (Ala62Val, Val75Ile, Phe77Leu, Phe116Tyr, Gln151Met) and mutations which confer resistance to nucleoside analogues and which should be reported for their potential relevance in treatment strategies (Met41Leu, Lys65Arg, Asp67Asn, Thr69Asp, Lys70Arg, Lys70Glu, Leu74Val, Val75Thr, Met184Val, Leu210Trp, Trp215Tyr, Thr215Phe, Lys219Gln).
For some subjects with very low plasma viral load at the end of treatment, the RT sequence could not be obtained from plasma. Sequences were then determined from proviral DNA extracted from PBMC. In these cases, the baseline genotype first obtained from plasma RNA was also obtained from proviral DNA.
All comparisons between groups were made with the Mann-Whitney non-parametric test using Statgraphic Plus version 3 (Manugistics, Rockville, Maryland, USA).
HIV-1 RT sequences from paired samples obtained at baseline and at the end of the follow-up were available for 39 patients. The last sample was obtained at W24, W32, W40, and W48 for 9, 2, 1, and 27 patients, respectively.
RT sequence analysis
At baseline, two subjects had a ZDV-related mutation: Leu210Trp (patient no. 37) and Asp67Asn plus Lys219Gln (patient no. 47). These mutations persisted at the end of the follow-up and additional mutations were detected at codons 215 (no. 37) or 70 (no. 47). Twenty-one of the 37 subjects without resistance mutations at baseline remained free of resistance mutations at the end of the follow-up (Group I), whereas 18 (46%) of the 39 subjects-including those with resistance mutations at baseline-developed one or more resistance mutations (Group II). Four patients from group I (nos 3, 17, 27, 43) presented a Gly196Glu substitution at baseline which persisted at the end of follow-up. This mutation was also detected in one patient (no 20) at the end of follow-up in association with a Lys70Arg mutation. This sporadically described mutation which occurs naturally in some patients  was not considered as a resistance mutation.
Genotypes at baseline and at the end of follow-up are given in Table 1. The ddI-specific mutation Leu74Val was not detected, whereas the d4T-specific mutation Val75Thr was found in patient no. 12 at W24. One or more ZDV-specific mutations appeared in 14 (36%) of the 39 subjects. Mutation at codon 215 was the most frequently detected [in nine (23%) patients]. The Gln151Met MDR mutation was found in four (10%) samples; this mutation was associated with the Phe116Tyr and Phe77Leu MDR mutations in one case (patient no. 36) and with the Met41Leu ZDV-related mutation in another (patient no. 23). Other mutations known to confer resistance to nucleoside analogues (Ala62Val, Lys65Arg, Lys70Glu, Val75Ile and Met184Val) were not detected.
Plasma viral load
Plasma viral loads (PVL) at baseline (median, 4.42log10 copies/ml) and at the end of follow-up (median, 3.27log10 copies/ml) are reported in Table 1. Median PVL value was 4.83log10 copies/ml in Group I and 4.93log10 copies/ml in Group II (P=0.37); median PVL value at the end of the follow-up was 2.70log10 copies/ml in Group I and 3.69log10 copies/ml in Group II (P=0.05). Median PVL decrease from baseline was higher in Group I than in Group II (-2.22log10 copies/ml versus -1.14log10 copies/ml; P=0.05).
The aim of the present study was to define the HIV-1 RT genotypic resistance patterns that developed in antiretroviral-naive subjects treated with d4T/ddI dual therapy. Our results clearly indicate that this combination administered for 24-48 weeks selects for the emergence of ZDV-resistance and MDR mutations. Indeed, one or more mutations at codons conferring ZDV resistance were developed by 36% of the patients, whereas the Gln151Met MDR mutation, alone or in combination with other ZDV-resistance or MDR mutations was developed in 10%. Resistance mutations reported as d4T-specific, such as Ile50Thr or insertions at codon 69 were not detected, whereas the Val75Thr mutation was developed by only one patient. We also did not detect the Leu74Val mutation known to confer resistance to ddI. These results are in agreement with data reported recently.
The occurrence of MDR mutations in patients treated with d4T/ddI combination therapy is not surprising as these mutations are associated with other nucleoside analogue combination therapies such as ZDV/ddI or ZDV/ddC. On the other hand, it has been reported that ZDV-resistance mutations could be selected by ddI monotherapy. Moreover, reduction in ZDV-susceptibility has been reported in ZDV-naive patients treated with d4T. Conversely, it was reported that HIV-1 with decreased susceptibility to d4T frequently harboured ZDV-resistance mutations. Therefore, in comparison with the wild-type virus, HIV-1 variants with ZDV-resistance mutations have probably a replicative advantage in the presence of d4T and ddI.
Although some patients (nos. 8, 25, 30) had a very low PVL in spite of ZDV-resistance or MDR mutations, it was observed that patients who developed mutations had a higher PVL at the end of the follow-up and showed a lower decrease in PVL in comparison with patients who remained free of resistance mutations. This observation could be explained by a decreased susceptibility of the HIV-1 variants to the d4T/ddI combination therapy. However, considering the limits of the genotyping test and the uncertainties about the impact of ZDV-specific and MDR mutations on d4T or ddI susceptibility, further investigations based on phenotypic analysis are needed to determine the susceptibility profile of the HIV-1 mutant selected by d4T/ddI combination therapy.
Appearance of ZDV-resistance or MDR mutations in patients treated with d4T/ddI would probably impair subsequent treatments with other nucleoside analogues, ZDV in particular. In the absence of genotypic or phenotypic susceptibility testing, it may be inappropriate to recommend ZDV in new antiretroviral regimens after d4T/ddI combination therapy failure. On the other hand, as emergence of RT mutations in these patients is a probable consequence of suboptimal treatment efficacy, the use of d4T/ddI combination should be only recommended in the context of highly active antiretroviral therapy.
In summary, we showed that d4T/ddI dual therapy selects ZDV-specific resistance and MDR mutations. These data should be taken into consideration in the management of the antiretroviral treatment after a d4T/ddI combination therapy failure.
1. Kellam P, Boucher CAB, Tijnagel JM, Larder BA. Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance. J Gen Virol
2. Tisdale M, Kemp SD, Parry NR, Larder BA. Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3‚-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci USA
3. Winters MA, Shafer RW, Jellinger RA, Mamtora G, Gingeras T, Merigan TC. Human immunodeficiency virus type 1 reverse transcriptase genotype and drug susceptibility changes in infected individuals receiving dideoxyinosine monotherapy for 1 to 2 years. Antimicrob Agents Chemother
4. Fitzgibbon JE, Howell RM, Haberzettl CA, Sperber SJ, Gocke DJ, Dubin DT. Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2‚-3‚ dideoxycytidine. Antimicrob Agents Chemother
5. Shirasaka T, Kavlick MF, Ueno T, et al. Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci USA
6. Deminie CA, Bechtold CM, Riccardi K, et al. Clinical HIV-1 isolates remain sensitive to stavudine following prolonged therapy. AIDS
7. Soriano V, Dietrich U, Villalba N, et al. Lack of emergence of genotypic resistance to stavudine after 2 years of monotherapy [letter]. AIDS
8. Lacey SF, Larder BA. Novel mutation (V75T) in human immunodeficiency virus type 1 reverse transcriptase confers resistance to 2‚, 3‚ didehydro-3‚-deoxythymidine in cell culture. Antimicrob Agents Chemother
9. Gu Z, Gao Q, Fang H, Parniak MA, Brenner BG, Wainberg MA. Identification of novel mutations that confer drug resistance in the human immunodeficiency virus polymerase gene. Leukemia
10. Whitcomb JM, Limoli K, Wrin T, et al. Phenotypic and genotypic analysis of stavudine-resistant isolates of HIV-1. Second International Workshop on HIV Drug Resistance, Treatment Strategies.
Lake Maggiore, Italy, June 1998 [abstract 17].
11. Reynes J, Denisi R, Bicart-Sée A, et al. STADI Pilot Study: once daily administration of didanosine (ddI) in combination with stavudine (d4T) in antiretroviral naive patients. 37th Interscience Conference on Antimicrobial Agents and Chemotherapy.
Toronto, September-October 1997 [abstract I-128A].
12. Segondy M, Izopet J, Pellegrin I, et al. Comparison of the QUANTIPLEX HIV-1 RNA 2.0 assay with the AMPLICOR HIV-1 MONITOR 1.0 assay for quantitation of levels of human immunodeficiency virus type 1 RNA in plasma of patients receiving stavudine-didanosine combination therapy. J Clin Microbiol
13. Schmit JC, Cogniaux J, Hermans P, et al. Multiple drug resistance to nucleoside analogues and nonnucleoside reverse transcriptase inhibitors in an efficiently replicating human immunodeficiency virus type 1 patient strain. J Infect Dis
14. Coakley E, Gillis J, Hammer S. Mutations in the reverse transcriptase (RT) coding region of HIV-1 isolates derived from individuals treated with didanosine and stavudine in combination. Second International Workshop on HIV Drug Resistance, Treatment Strategies.
Lake Maggiore, Italy, June 1998 [abstract 96].
15. Lin PF, Samanta H, Rose RE, et al. Genotypic and phenotypic analysis of human immunodeficiency virus type 1 isolates from patients on prolonged stavudine therapy. J Infect Dis
16. Bloor S, Hertogs K, Desmer RL, Pauwels R, Larder BA. Virological basis for HIV-1 resistance to stavudine investigated by analysis of clinical samples.
Second International Workshop on HIV Drug Resistance, Treatment Strategies.
Lake Maggiore, Italy, June 1998 [abstract 15].
Members of the STADI group: R. Denisi, M. Dupon, H. Fleury, J.-Y. Lacut, B. Leng, I. Pellegrin, J.-L. Pellegrin, J.-M. Ragnaud, Bordeaux University Hospital; A. Bicart-See, E. Bonnet, J. Izopet, B. Marchou, P. Massip, M. Obadia, J. Puel, Toulouse University Hospital; B. Delmas, J. Fabre, C. Favier, C. Leclercq, B. Montes, J. Reynes (principal investigator), M. Segondy, Montpellier University Hospital. Cited Here...
Zidovudine; stavudine; lamivudine; HIV-1; reverse transcriptase; resistance mutation
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