Chetchotisakd et al. Materials and Methods

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1 High Rate Multiple Drug Resistances in HIV-Infected Patients Failing Nonnucleoside Reverse Transcriptase Inhibitor Regimens in Thailand, Where Subtype A/E Is Predominant Ploenchan Chetchotisakd, MD, Siriluck Anunnatsiri, MD, Sasisopin Kiertiburanakul, MD, Ruengpung Sutthent, PhD, Thanomsak Anekthananon, MD, Chureeratana Bowonwatanuwong, MD, Boonchai Kowadisaiburana, MD, Khuanchai Supparatpinyo, MD, Manassinee Horsakulthai, MS, Sanchai Chasombat, MD, Kiat Ruxrungtham, MD, and the Study Team The prevalence of drug resistance was determined among 64 HIV-infected Thai patients who were failed while receiving nonnucleoside reverse transcriptase inhibitor (NNRTI) based regimens. Eighty-nine percent of patients had 1 or more NNRTI mutation resistances. Almost all patients had resistance to at least 1 nucleoside reverse transcriptase inhibitor (NRTI), and 42% had multiple-nrti resistance. Keywords: drug resistance in Thailand; NNRTI resistance; NRTI resistance; NNRTI failure Since the introduction of antiretroviral therapy for persons with HIV infection, the rates of mortality and morbidity related to HIV disease have decreased. 1 Current antiretroviral treatment guidelines recommend commencing a combined antiretroviral therapy when a patient has a CD4 count <200 to 350 cells/mm 3 or becomes symptomatic. The preferred first-line combinations are 2 nucleoside reverse transcriptase inhibitors (NRTIs) as a backbone plus either 1 nonnucleoside reverse transcriptase inhibitor (NNRTI) or 1 boosted protease inhibitor (PI). 2,3 Because of the cost and technical issues associated with viral load testing, it is not part of the World Health Organization (WHO) guidelines for antiretroviral therapy (ART) in resource-limited settings. 4 Thus, treatment failure is defined by clinical and immunological criteria 4 ; however, by the time clinical and/or immunological failure is recognized, most patients may have failed virologically for months or even years. Consequently, cumulative resistance-associated mutations will lead to high levels of cross-resistance within the antiretroviral class. There are no data on NNRTI drug resistance with DOI: / Sage Publications Please visit the Journal at the HIV clade A/E, which is the predominant clade in Thailand. Here we report the genotypic resistance pattern in patients with NNRTI-based regimen failure in routine clinical care from various hospitals in Thailand. Materials and Methods Sixty-four HIV-infected adults who had treatment failure after being treated with NNRTI-based regimens for at least 3 months were recruited. Treatment failure is defined as (1) virological failure (ie, the patients had viral load >1000 copies/ml after 3 months of treatment); (2) immunological failure (ie, the CD4 count decreased >30% of the maximum values); and/or (3) clinical failure (ie, the patients developed new or recurrent opportunistic infection after 6 months of therapy). Patients were enrolled between August 2003 and February 2004 from 7 hospitals in Thailand: Srinagarind Hospital, Khon Kaen University (n = 9); Chulalongkorn University Hospital (n = 10); Siriraj Hospital, Mahidol University (n = 11); Ramathibodi Hospital, Mahidol University (n = 9); Bamrasnaradura Institute (n = 9); Chiang Mai University Hospital (n = 10); and Chonburi Hospital (n = 6). The institutional From Srinagarind Hospital, Khon Kaen University, Khon Kaen (Chetchotisakd, Anunnatsiri, and Horsakulthai); Ramathibodi Hospital, Mahidol University, Bangkok (Kiertiburanakul); Siriraj Hospital, Mahidol University, Bangkok (Sutthent and Anekthananon); Chonburi Hospital, Chonburi (Bowonwatanuwong); Bamrasnaradura Institute, Nonthaburi (Kowadisaiburana); Chiang Mai University Hospital, Chiang Mai University, Chiang Mai (Supparatpinyo); Bureau of AIDS, TB, and STI, Department of Disease Control, Ministry of Public Health, Thailand (Chasombat); Chulalongkorn University Hospital, Chulalongkorn University; and the HIV-NAT, Thai Red Cross AIDS Research Centre, Bangkok (Ruxrungtham). There are no financial associations of conflicts of interest. Correspondence: Ploenchan Chetchotisakd, Srinagarind Hospital, Khon Kaen University, Khon Kaen 40002, Thailand; ploencha@kku.ac.th. 152 J INT ASSOC PHYSICIANS AIDS CARE 5(4); 2006 pp

2 High Rate Multiple Drug Resistances review board committees of the Thai Ministry of Public Health and each university approved the study. Patients signed a written informed consent. The following were done for each patient: (1) a complete blood cell and CD4 count; (2) an HIV RNA polymerase chain reaction (PCR) with Cobas Amplicor HIV-1 Monitor test, version 1.5 (Roche, Germany); and (3) an HIV-1 genotypic drug resistance assay using the TRUGENE HIV-1 genotyping kit (Visible Genetics, Toronto, Calif). The determination of NRTI mutations was based on the guidelines published by the International AIDS Society United States (IAS-USA) HIV Resistance Testing Guideline Panel Nucleoside analog resistance mutation (NAM) is defined by the presence of any of the following mutations: M41L, E44D, D67N, K70R, V118I, L210W, T215Y/F, or K219Q/E. A greater number of codon mutations (ie, 4) has been found to be associated with cross-resistance with other NRTI except lamivudine (3TC). Resistance to a specific NRTI is defined by the presence of mutations that cause resistance to that particular NRTI. The NNRTI-resistance mutation is defined by the presence of any of the following mutations: L100I, K103N, V106M, Y 181C/I, Y188L, G190S/A, or M230L. Statistical Analysis Descriptive analysis was performed to show the proportions of patients with various NRTI-specific, NNRTIspecific, and multidrug resistance. Factors including the HIV staging, CD4 count, HIV-RNA level, duration of prior ART, NRTI regimen (zidovudine [ZDV], 3TC, didanosine [ddi], or stavudine [d4t]), and prior dual NRTI experience were explored using univariate and multivariate logistic regression (backward logistic regression) analysis to evaluate the association between these factors and any multiple-nrti resistance. Results Of the 64 patients enrolled, 2 were nonevaluable because their samples were not amplifiable by PCR; thus, 62 patients were included in the analysis. The demographic characteristics, HIV disease status, ART data of the cohort, and criteria for treatment failure are presented in Table 1. The majority of patients were AIDS with a median CD4 of 105 cells/mm 3 and a mean HIV-RNA of 4.45 log 10 copies/ml. Most of the patients were receiving their first treatment of an NNRTI-based regimen, which included 3TC, d4t, and nevirapine (NVP). However, 16 patients (26%) received dual NRTI regimens before NNRTI-based regimens. Nevirapine was the most commonly used NNRTI drug (69%). The NRTI backbone for most patients was d4t/3tc (55%), followed by ZDV/ddI Table 1. Demographic Characteristics and HIV Disease Status and Antiretroviral Therapy (ART) Data for a Cohort of 62 HIV-Infected Thai Patients at the Enrollment Value Characteristic n = 62 Age, mean years (SD) 34.9 (6.0) Sex, n (%) Male 36 (58) Female 26 (42) Risk factor of HIV infection, n (%) Heterosexual 58 (94) Intravenous drug abuser 4 (6) HIV disease status, n (%) A 18 (29) B 6 (10) C 38 (61) Median CD4 count, cells/mm 3 (IQR) 105 (30-184) Mean HIV-1 RNA level, log 10 copies/ml (SD) 4.45 (0.81) First ART treatment, n (%) NNRTI-based regimen 46 (74) Prior dual NRTI regimen 16 (26) Type of NNRTI used, n (%)* Nevirapine 43 (69) Efavirenz 21 (34) Type of NRTI used, n (%) Lamivudine 51 (82) Stavudine 49 (79) Zidovudine 21 (34) Didanosine 23 (37) Duration of ART, median weeks (IQR) 145 (84-192) Criteria for treatment failure, n (%) Virological failure 40 (64) Immunological failure 19 (31) Clinical failure 3 (5) IQR, interquartile range; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; ART, antiretroviral therapy. *2 patients had history of previous different NNRTI-based regimen. (17%), ZDV/3TC (13%), d4t/ddi (13%), and ddi/3tc (1%). The median duration of ART was 145 weeks (interquartile range ). The majority of patients experienced treatment failure, as defined by virological criteria (64%). The prevalence of HIV with NNRTI resistance mutations is presented in Table 2. Most of the patients (89%) had 1 or more mutations associated with NNRTI resistance. Mutations at G190S/A and Y181C/I were the most common sites. No mutations at L100I, V106M, and M230L were detected. Mutation at K103N was significantly associated with EFV use, and Y181C/I was associated with NVP use (P =.001). The prevalence of HIV with NRTI resistance is presented in Table 2. Almost all (95%) of patients had at least 1 NRTI resistance associated mutation. The most frequently observed amino acid change was M184V/I, which confers resistance to 3TC (38 of 51 cases exposed to 3TC). Seventy-three percent had at least 1 NAM. Among these mutations, D67N (n = 34) J INT ASSOC PHYSICIANS AIDS CARE 5(4);

3 Table 2. Number of Patients With Specific Mutations by Drug Class and Nonnucleoside Reverse Transcriptase Inhibitor (NNRTI) Mutation Comparison Between Nevirapine (NVP)-Based and Efavirenz (EFV)-Based Groups NVP-Based EFV-Based Group Overall Group (n = 42) (n = 20) Mutation No. of Patients (%) No. of Patients (%) No. of Patients (%) P Value NNRTI mutation Any NNRTI 55 (89) 36 (86) 19 (95).41 K103N 19 (31) 7 (17) 12 (60).001 Y181C/I 25 (40) 23 (55) 2 (10).001 Y188L 8 (13) 6 (14) 2 (10) 1.00 G190S/A 27 (44) 19 (45) 8 (40).70 1 NNRTI mutation 34 (55) 19 (45) 15 (75).03 >1 NNRTI mutations 21 (34) 17 (41) 4 (20).11 NRTI mutation Any NRTI mutation 59 (95) M41L 21 (34) E44D 5 (8) K65R 11 (18) D67N 34 (55) K70R 20 (32) L74V 3 (5) V118I 13 (21) M184V/I* 38 (61) L210W 17 (27) T215Y/F 25 (40) K219Q/E 17 (27) Q151M complex 7 (11) Any NAM 45 (73) 4 NAMs 20 (32) 4 NAMs and/or Q151M 26 (42) NAM, nucleoside analog resistance mutation. NAMs include M41L, E44D, D67N, K70R, V118I, L210W, T215Y/F, and K219Q/E mutations. *75% (38/51 cases) among lamivudine-experienced patients. % of patients No mutation Figure NNRTI 61 3TC 95 Any NRTI was the most common, followed by T215Y/F, M41L, and K70R, respectively. Thirty-two percent of the 73 Any NAMs 32 >/=4NAMs 11 Q151M 42 MDR-NRTI Prevalence of HIV with nonnucleoside reverse transcriptase inhibitor (NNRTI) and nucleoside reverse transcriptase inhibitor (NRTI) resistance among 62 HIV-infected patients. NNRTI-resistance mutation included L100I, K103N, V106M, Y 181C/I, Y188L, G190S/A, and M230L. Nucleoside analog resistance mutations (NAMs) included M41L, E44D, D67N, K70R, V118I, L210W, T215Y/F, and K219Q/E mutations. 3TC, lamivudine-resistance mutations (M184V/I) and Q151M, the multidrug-resistance mutation. MDR- NRTI, the multiple-nrti-drug resistance, the combination of patients with 4 NAMs and/or Q151M. patients had 4 NAMs. Only 3 patients had a didanosinespecific mutation (L74V). None of the patients had HIV with multiple-nrti-resistance 69-insertion complex. When patients with 4 NAMs and/or Q151M were considered, 42% had multiple-nrti resistance. M184V/ I mutation was found only in patients who had received 3TC. Seven patients had Q151M multidrug-resistance mutation. The prevalence of HIV with NNRTI, 3TC, NRTI, and multidrug-resistance-nrti among 62 HIVinfected patients is summarized in Figure 1. In multivariate analysis, adjusted for HIV staging, the HIV RNA level, the CD4 count, the duration of treatment with antiretroviral (ARV) medications, NRTI regimens, and prior dual NRTI experience, only ddi exposure was significantly related to Q151M mutation (odds ratio [OR] 22.82; 95% confidence interval [CI] ; P =.01). The presence of 4 NAMs was significantly related to the duration of ARV medication exposure >96 weeks (OR 5.54; 95% CI ; P =.035), advances HIV infection (OR 5.54; 95% CI ; P =.035), and high baseline viral load (OR 2.84; 95% CI ; P =.01). In multivariate analysis, adjusted for HIV staging, the HIV RNA level, CD4 count, duration of ART, NRTI regimens, and prior dual NRTI experience, 154 J INT ASSOC PHYSICIANS AIDS CARE 5(4); 2006

4 High Rate Multiple Drug Resistances only the duration of ARV medication exposure >96 weeks (OR 6.29; 95% CI ; P =.042) and high baseline HIV-RNA (OR 3.92; 95% CI ; P =.011) were significantly associated with the presence of 4 NAMs. Discussion Our study shows that the majority of patients who experienced treatment failure while receiving NNRTIbased regimens had HIV with both NNRTI and NRTI mutations. Eighty-nine percent of the patients had NNRTI mutations, which conferred resistance to the entire NNRTI drug class. Among the NRTI mutations, 61% had M184V and 73% had HIV with NAMs, which conferred cross-resistance among NRTIs. Infection with HIV with 4 NAMs and Q151M was associated with resistance to all NRTIs. Forty-two percent of the patients in our study had HIV with 4 NAMs and/or Q151M, and, as a consequence, the likelihood of treatment with new NRTIs having adequate viral suppressive effect was poor. In addition, in the majority of patients receiving 3TC-based regimens (75%), mutations conferring 3TC resistance were very common, further limiting the choice of NRTIs. It is unlikely that such patients can be salvaged with the 2 new NRTIs plus boosted PI, as recommended by the guidelines. 6 Our study also confirmed other reports that crossresistance to both ZDV and d4t, by selecting and accumulating NAMs, is almost universal Interestingly, 3 of our patients with the L74V mutation, which conferred resistance to ddi, did not receive ddi as part of the treatment regimen before (data not shown). However, the use of ddi was associated with Q151M, a multidrugresistant mutation, and HIV with the multiple-nrtiresistance 69-insertion complex was not detected. These findings concur with 2 other Thai studies. 8,10 Sixteen patients in the cohort were switched from dual NRTIs (ie, ZDV/ddI, ZDV/3TC, d4t/3tc, or d4t/ddi) to NNRTI-based regimens. Among these patients, NAMs might have already been selected before switching, 7-10 resulting in rapid virological failure attributable to the emergence of NNRTI resistance. A fixed-dose combination of d4t/3tc/nvp was the most commonly used in this cohort. Evidence indicates that NNRTI resistance tends to develop during drug holiday because NVP and EFV have a much longer halflife than NRTIs, resulting in a de facto monotherapy. 11 Lamivudine resistance will consequentially be developed after reinitiation of the medication because the regimen relies on dual NRTIs. As we demonstrated, the longer patients are on this inadequate regimen, the greater the accumulation of NAMs. As evidenced by our multivariate analysis, patients who experienced treatment for longer than 96 weeks were associated with the presence of 4 NAMs (OR 6.29; 95% CI ; P =.042). The NRTI mutation seen in this cohort was not different from mutations in patients infected with the HIV clades A, B, C, and F. 8-10,12,13 Of interest, for NNRTI mutations, the most common mutation was G190S/A. By comparison, with the HIV clades A, C, and D, the most common mutation was K103N. 14,15 However, we found that K103N was associated with EFV use and Y181C/I was associated with NVP use similar to HIV clade B. 15 Fixed-dose combinations of 3TC/d4T/NVP and ZDV/3TC/NVP are currently available in Thailand. In our study, a fixed-dose combination of 3TC/d4T/NVP called GPO-VIR was the commonly used. Similar fixeddose combinations of these drugs are also available in Africa and India. These drugs potentially serve as a prime choice for ART in resource-limited settings because of the low price, low drug interaction, and low incidence of long-term side effect on lipids. NVP demonstrated a similar efficacy compared with EFV in the 2NN Study. 16 Early cutaneous side effects and hepatotoxicity can be managed by careful monitoring and initiation with lead-in fashion. Hepatoxicity is less common in patients with low CD4 count, which are the major population of ART initiators in resourcepoor settings. However, combining drugs that have a low resistance threshold, such as 3TC and NVP, would likely result in treatment failure among noncompliant patients. With increasing use of ART, in accordance with the WHO s 3 by 5 Initiative Campaign, more patients with drug resistance are inevitable, especially without viral load testing. The present study provides an important message on the high prevalence of HIV with NNRTI and 3TC resistance mutations in patients treated with NNRTIbased regimens that include 3TC as the NRTI backbone; we also demonstrated the high prevalence of 4 NAMs in patients who had been on ARV medication for longer than 96 weeks and those who had a high baseline viral load. Hence, there are very limited options regarding new and effective NRTIs for the salvage regimen in these situations. Affordable and appropriate salvage regimens for patients who fail NNRTIbased therapy in resource-limited countries need further investigation in a large clinical trial. Therefore, wide implementation of effective and practical measures to encourage treatment adherence is the most critical challenge for this important global treatment access program. Aggressive effort is needed to make a low-cost viral load assay available for resource-poor settings, to enable early detection of virological failure, and to prevent development of cross-resistance viruses. J INT ASSOC PHYSICIANS AIDS CARE 5(4);

5 Acknowledgments We thank the patients for their participation, professors Surapol Suwannakul and Praphan Phanuphak for their advice and support, and Mr. Bryan Roderick Hamman for assistance with the English-language presentation. Study Team Piroon Mootsikapun and Parichat Seawsirikul, Srinagarind Hospital, Khon Kaen University, Khon Kaen; Bucha Piyavong, Ramathibodi Hospital, Mahidol University, Bangkok; Winai Ratanasuwan, Wichai Techasathit, Nitaya Chuengprasert, and Daungnapha Aeworn, Siriraj Hospital, Mahidol University, Bangkok; Nophanath Chumpathat, Patamavadee Termvises, and Suphida Thongyen, Bamrasnaradura Institute, Nonthaburi; Romanee Chaiwarith, Chiang Mai University Hospital, Chiang Mai University, Chiang Mai; Somyot Kittimunkong, Bureau of AIDS, TB, and STI, Department of Disease Control, Ministry of Public Health, Thailand; and Chris Dumcomb and Jiranan Sewatatat, HIV-NAT, Thai Red Cross AIDS Research Centre, Bangkok. Financial Support Provided through a grant from the Bureau of AIDS, TB, and STI, Department of Disease Control, Ministry of Public Health, Thailand. Potential Conflicts of Interest Kiat Ruxrungtham and Thanomsak Anekthananon received research grant support from the Government Pharmaceutical Organization (GPO), which manufactures GPO VIR. 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: Gazzard B, BHIVA Writing Committee. British HIV Association (BHIVA) guidelines for the treatment of HIV-infected adults with antiretroviral therapy (2005). HIV Med. 2005;6(suppl 2): Guidelines for the use of antiretroviral agents in HIV-1- infected adults and adolescents. DHHS Panel on Clinical Practice for Treatment of HIV infection. Available at: Accessed April 7, WHO. Scaling up antiretroviral therapy in resource-limited settings: treatment guidelines for a public health approach. Geneva: WHO; Johnson VA, Brun-Vezinet F, Clotet B, et al. Update of the drug resistance mutations in HIV-1: Top HIV Med. 2005; 13: Yeni PG, Hammer SM, Hirsch MS, et al. Treatment for adult HIV infection: 2004 recommendations of the International AIDS Society-USA Panel. JAMA. 2004;292: Maxeiner HG, Keulen W, Schuurman R, et al. Selection of zidovudine resistance mutations and escape of human immunodeficiency virus type 1 from antiretroviral pressure in stavudine-treated pediatric patients. J Infect Dis. 2002;185: Sirivichayakul S, Ruxrungtham K, Ungsedhapand C, et al. Nucleoside analogue mutations and Q151M in HIV-1 subtype A/E infection treated with nucleoside reverse-transcriptase inhibitors. AIDS. 2003;17: van Rossum AM, Fraaij PL, de Groot R. Efficacy of highly active antiretroviral therapy in HIV-1-infected children. Lancet Infect Dis. 2002;2: Lolekha R, Siangphoe U, Pancharoen C, et al. Resistance to dual nucleoside reverse transcriptase inhibitors in children infected with HIV clade A/E. Clin Infect Dis. 2005;40: Taylor S, Allen S, Fidler S, et al. Stop study: after discontinuation of efavirenz, plasma concentrations may persist for 2 weeks or longer. Paper presented at: 11th CROI; February 2004; San Francisco, Calif. 12. de Mendoza C, Ramos JT, Ciria L, et al. Efficacy and safety of stavudine plus didanosine in asymptomatic HIV-infected children with plasma HIV RNA below 50,000 copies per milliliter. HIV Clin Trials. 2002;3: Pellegrin I, Izopet J, Reynes J, et al. Emergence of zidovudine and multidrug-resistance mutations in the HIV-1 reverse transcriptase gene in therapy-naive patients receiving stavudine plus didanosine combination therapy. STADI Group. AIDS. 1999;13: Eshleman SH, Hoover DR, Chen S, et al. Nevirapine (NVP) resistance in woman with HIV-1 subtype C, compared with subtypes A and D after the administration of single-dose NVP. J Infect Dis. 2005;192: Grossman Z, Istomin V, Averbuch D, et al. Genetic variation at NNRTI resistance-associated positions in patients infected with HIV-1 subtype C. AIDS. 2004;18: van Leth F, Phanuphak P, Ruxrungtham K, et al. Comparison of first-line antiretroviral therapy with regimens including nevirapine, efavirenz, or both drugs, plus stavudine and lamivudine: a randomised open-label trial, the 2NN Study. Lancet. 2004;363: J INT ASSOC PHYSICIANS AIDS CARE 5(4); 2006