World Health Organization (WHO) guidelines recommend a combination of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) as a first-line regimen in resource-poor settings to treat HIV-1 infection. Currently, only one WHO prequalified combination of NRTI/NNRTI is available as a fixed-dose combination, combining stavudine, lamivudine and nevirapine, and its use has been shown to be effective [1–3]. There is, however, a lack of information on the resistance patterns with stavudine/lamivudine/nevirapine fixed-dose combination at failure. As genotypic tests are not widely available in resource-poor countries, it is important to characterize the resistance patterns at failure with this kind of regimen in the setting of second-line therapy. As part of a simplified strategy for providing HAART in resource-poor settings, the Initiative Malienne d'Accès aux Antirétroviraux recommends the use of stavudine/lamivudine/nevirapine fixed-dose combination (Triomune) in its HIV/AIDS programme whenever possible. These drugs are currently prescribed free of charge in the majority of newly treated patients. We report here the experience with the use of stavudine/lamivudine/nevirapine in a programme implemented by Solidarité Thérapeutique et Initiatives contre le SIDA (SOLTHIS) in Ségou, Mali. SOLTHIS is a non-governmental organization involved in HIV/AIDS. The objective of SOLTHIS is to improve the access to antiretroviral drugs for people living with HIV/AIDS in resource-poor countries. The objective of this study was to characterize the resistance patterns at the time of failure after a first-line regimen containing stavudine/lamivudine/nevirapine as a fixed-dose combination in Mali.
A total of 109 HIV-1-infected patients who started stavudine/lamivudine/nevirapine as an initial regimen between January 2004 and August 2005 were included in this study. The dosing of stavudine was 30 mg for all patients using the appropriate fixed-dose combination. Women who had previously received HIV vertical transmission prophylaxis were excluded from the study. Clinical assessment was planned at baseline and monthly, CD4 cell counts were evaluated every 6 months. HIV-1 RNA was quantified by Roche Amplicor assay (version 1.5; Roche Diagnostic Systems Inc., Branchburg, New Jersey, USA) after a minimum of 6 months of HAART. In the case of an HIV-1-RNA level greater than 200 copies/ml, a resistance genotype was performed by automated population-based full-sequence analysis (ABI system) using the Agence Nationale de Recherches sur le SIDA consensus technique . Resistance mutations were recorded according to the 2006 International AIDS Society list of mutations . Nevirapine plasma concentrations were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection after liquid extraction on the plasma used for the determination of the viral load [6,7]. The nevirapine assay was validated over concentration ranges of 50–10 000 ng/ml and the limit of quantification was 50 ng/ml. On the basis of previous findings, the nevirapine plasma concentration was considered adequate if it reached 3400 ng/ml and the therapeutic range was 3400–6000 ng/ml. For statistical analyses, comparisons between groups were made using the Mann–Whitney test for quantitative variables and the chi-squared test for qualitative variables. Statview software version 5 was used (SAS Institute Inc., Cary, North Caroline, USA).
Of the 109 patients in the study, 61% were women. At baseline, the median age was 35 years, median weight was 48 kg, median CD4 cell count was 105 cells/μl, and 93% of patients were WHO stage III–IV. At a median time of 6.7 months after initiation, 83 patients (76%) had HIV-1-RNA levels of less than 200 copies/ml. Among the 26 patients with detectable HIV-1 RNA (median 4150 copies/ml; range 209–404 000 copies/ml), viruses were not amplifiable in four cases. The 22 remaining patients harboured viruses with no mutation in 11 cases and resistance mutations in 11 cases: one M184V alone, one Y181C alone, nine M184V plus NNRTI mutations (five Y181C, two K103N, one V106A and three G190A; Table 1). No thymidine analogue mutations (TAM) were observed. All sequenced viruses were CRF_02 AG subtype. The plasma nevirapine concentration was measured for 93 patients (median 7424 ng/ml; range 50–35 499 ng/ml). According to the plasma nevirapine concentration, patients were classified as non-adherent (5.4% with nevirapine < 50 ng/ml), below the therapeutic range (9.7% with nevirapine < 3400 ng/ml), and above the therapeutic range (34.4% with nevirapine > 8000 ng/ml). Patients with virological failure harboured a lower median plasma nevirapine concentration (6409 ng/ml, mean 5806, range 50–14 085) than patients with virological success (7616 ng/ml, mean 8706, range 495–35 499).
In this study, early failures (median time 6 months) to stavudine/lamivudine/nevirapine were associated in 50% of cases with no resistance mutation. This suggests that, as in developing countries, poor adherence can be responsible for some cases of virological failure. More than 50% of the viruses harbouring NNRTI mutations contained the Y181C mutation. This should be further explored and could be related to the use of stavudine, which would not prevent the selection of the Y181C mutation, in contrast to what was observed in subtype B when zidovudine was associated with nevirapine or to the CRF_02 subtype that is predominant here, as it has been shown that the HIV-1 subtype can influence the type of nevirapine mutation (higher frequency of Y181C for subtype A and K103N for subtype D) [8–10]. The absence of TAM is in accordance with previous results showing a low rate of TAM selection when lamivudine is combined with zidovudine or stavudine, probably in relation to the interaction between M184V and T215Y/F mutations . The presence of the M184V mutation in most cases, which appears to delay or prevent the emergence of TAM, combined with the fact that sequences have been made early, can explain this result. The absence of TAM suggests that most NRTI can be used in a second-line regimen after early failure to stavudine/lamivudine/nevirapine. These results reinforce the importance of the accessibility of HIV-RNA assays for monitoring treated patients in resource-poor countries, where resistance testing is not available, to detect early virological failure in order to preserve future therapeutic options. In the case of prolonged viral replication under treatment, resistance mutations can emerge and the late detection of virological failure can limit the use of NRTI in a second-line regimen. It is thus crucial to expand the accessibility of virological testing (HIV-RNA assays, resistance tests) concomitantly with the accessibility of antiretroviral treatment.
The authors would like to thank the Fondation Bettencourt Schueller.
Sponsorship: This study was supported by Solidarité Thérapeutique et Initiatives contre le SIDA (SOLTHIS).
This work was presented at the XVth International HIV Drug Resistance Workshop, 13–17 June 2006, Sitges, Spain (abstract 77), and was also presented at the 14th Conference on Retroviruses and Opportunistic Infections, 25–28 February 2007, Los Angeles, USA.
1. Bourgeois A, Laurent C, Mougnutou R, Nkoué N, Lactuock B, Caffi L, et al
. Field assessment of generic antiretroviral drugs: a prospective cohort study in Cameroon. Antivir Ther 2005; 10:335–341.
2. Calmy A, Pinoges L, Szumilin E, Zachariah R, Ford N, Ferradini L. Generic fixed-dose combination antiretroviral treatment in resource-poor settings: multicentric observational cohort. AIDS 2006; 20:1163–1169.
3. Laurent C, Kouanfack C, Koulla-Shiro S, Nkoué N, Bourgeois A, Calmy A, et al
. Effectiveness and safety of a generic fixed-dose combination of nevirapine, stavudine, and lamivudine in HIV-1-infected adults in Cameroon: open-label multicentre trial. Lancet 2004; 364:29–34.
4. Descamps D, Delaugerre C, Masquelier B, Ruffault A, Marcelin AG, Izopet J, et al
. Repeated HIV-1 resistance genotyping external quality assessments improve virology laboratory performance. J Med Virol 2006; 78:153–160.
5. Johnson VA, Brun-Vezinet F, Clotet B, Kuritzkes DR, Pillay D, Schapiro JM, Richman DD. Update of the drug resistance mutations in HIV-1: fall 2006. Top HIV Med 2006; 14:125–130.
6. Martinez V, Marcelin AG, Morini JP, Deleuze J, Krivine A, Gorin I, et al
. HIV-1 intermittent viraemia in patients treated by nonnucleoside reverse transcriptase inhibitor-based regimen. AIDS 2005; 19:1065–1069.
7. Wirden M, Simon A, Schneider L, Tubiana R, Paris L, Marcelin AG, et al
. Interruption of nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy for 2 months has no effect on levels of human immunodeficiency virus type 1 in plasma of patients harboring viruses with mutations associated with resistance to NNRTIs. J Clin Microbiol 2003; 41:2713–2715.
8. Delaugerre C, Rohban R, Simon A, Mouroux M, Tricot C, Agher R, et al
. Resistance profile and cross-resistance of HIV-1 among patients failing a nonnucleoside reverse transcriptase inhibitor-containing regimen. J Med Virol 2001; 65:445–448.
9. Eshleman SH, Guay LA, Wang J, Mwatha A, Brown ER, Musoke P, et al
. Distinct patterns of emergence and fading of K103N and Y181C in women with subtype A vs. D after single-dose nevirapine: HIVNET 012. J Acquir Immune Defic Syndr 2005; 40:24–29.
10. Richman DD, Havlir D, Corbeil J, Looney D, Ignacio C, Spector SA, et al
. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J Virol 1994; 68:1660–1666.
11. Mouroux M, Descamps D, Izopet J, Yvon A, Delaugerre C, Matheron S, et al
. Low-rate emergence of thymidine analogue mutations and multidrug resistance mutations in the HIV-1 reverse transcriptase gene in therapy-naive patients receiving stavudine plus lamivudine combination therapy. Antivir Ther 2001; 6:179–183.