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Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART
Journal of Clinical Virology 25 (2002) 39 – 46 www.elsevier.com/locate/jcv
Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART Amilcar Tanuri a,*, Elena Caridea a, Maria C. Dantas b, Marisa G. Morgado c, Daise L.C. Mello c, Sandra Borges j, Marisa Tavares d, Selma B. Ferreira d, Guilherme Santoro-Lopes d, Claudia R.F. Martins f, Andre´ L.C. Esteves f, Ricardo S. Diaz h, Sandra M.S. Andreo h, Luiz A.P. Ferreira k, Rodrigo Rodrigues e, Tania Reuter e, Ana M.S. Cavalcanti g, Suelene M. de Oliveira g, Heraclito B. de Barbosa i, Paulo R. Teixeira b, Pedro N. Chequer b a
Departamento de Gene´tica, Laborato´rio de Virologia Molecular, Instituto de Biologia, Uni6ersidade Federal do Rio de Janeiro, CCS, Bloco A, sala 121, 2o andar, Rio de Janeiro, RJ, Cep: 21944 -970,. Brazil b Programa Brasileiro de AIDS/STD, Brazilian Ministry of Health, Brasilia, Brazil c Laborato´rio de Imunologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil d Laborato´rio Petrobras, Hospital Uni6ersita´rio Clementino Fraga Filho, UFRJ, Rio de Janeiro, Brazil e Nucleo de Doenc¸as Infeciosas, Uni6ersidade Federal do Espirito Santos, Espirito Santos, Brazil f Laborato´rio de Virologia, Uni6ersidade de Brası´lia, Brasilia, Brazil g Laborato´rio Central de Sau´de Pu´blica, Recife, Pernambuco, Brazil h Laborato´rio de Retro6irologia, Uni6ersidade Federal do Estado de Sa˜o Paulo, Sao Paulo, Brazil i Departamento de Epidemiologia, Uni6ersidade de Sa˜o Paulo, Sao Paulo, Brazil j Laborato´rio Central de Sau´de Pu´blica, Florianopolis, Santa Catarina, Brazil k Uni6ersidade Federal de Santa Catarina, Florianopolis, Santa Catarina, Brazil Received 11 July 2001; accepted 20 November 2001
Abstract Background: Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. Objecti6e: In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. Study Design: Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations
* Corresponding author. Tel.: + 55-21-280-8043; fax: + 55-21-280-0994 E-mail address: [email protected] (A. Tanuri). 1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 1 ) 0 0 2 4 9 - 9
40
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
related to drug resistance in RT gene was analyzed using a line probe assay (LIPA®) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. Results: There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. Conclusions: This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HIV-1; Protease; Reverse transcriptase; Phenotyping; Genotyping
1. Introduction Recent development of a new generation of drugs against HIV has led to marked changes in antiretroviral (ARV) therapy and treatment of HIV infection. ARV drug combinations including two nucleoside reverse transcriptase (RT) inhibitors and/or non-nucleoside RT inhibitors can reduce the viral load in plasma to undetectable levels and provide durable clinical benefits (Vella et al., 1996; Zhang et al., 1999). However, complete suppression of HIV-1 replication is rarely achieved with mono or dual therapy, which have a transient effect favoring the development of ARV resistance and cross resistance. For this reason, current guidelines for ARV therapy recommend at least triple-drug combination, the socalled highly active antiretroviral therapy (HAART). However, development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV (Shafer et al., 1998; Ledergerber et al., 1999). Continuous in vivo HIV replication and the error prone nature of the viral RT are the driving forces for generation of drug resistance (Hu et al., 1996). Moreover, recent studies identified replication-competent HIV-1 reservoirs in patients under HAART, raising a serious concern as to whether antiviral therapy can eradicate HIV-1 (Finzi et al., 1997; Zhang et al., 1999). The appearance of viruses resistant to ARV drugs is one of the reasons for therapeutic failure, characterized broadly as inadequate viral suppression, unsatisfactory increase in CD4+ T-cell counts, or clinical progression. This failure may be associated with other factors such as patient adherence, pharmacological factors or emergence
of resistant viruses. Moreover, there is increasing concern due to their further transmission of these resistant strains (Gomez-Cano et al., 1998; Tanuri et al., 1999b; Brindeiro et al., 1999). The Brazilian Ministry of Health (MOH) has been sponsoring a policy of universal access to ARV drugs for AIDS patients since 1996. By January 2001, Brazil had about 100 000 patients using these drugs in the Public Health System. The Brazilian MOH has adopted periodically reviewed guidelines to ARV treatment, in order to facilitate adequate and uniform prescription all over the country, as well as guarantee a more rational and logistical approach of the use of these drugs in the Public Health System. Moreover, the Brazilian MOH implemented a network of 70 laboratories distributed throughout the country to quantify viral load and measure CD4 + T-cell count in every HIV-infected individual treated in a public institution, allowing better monitoring of these individuals. The percentage of these patients experiencing virological failure related to drug-resistant strains is largely unknown. These patients could be a source of transmission of these variants to the recently infected individuals, starting a new epidemic wave. This work describes the ARV resistance profile in patients failing HAART in Brazil, in an attempt to correlate the HIV therapeutic failure with RT and protease resistance mutations.
2. Materials and methods This study was approved by Brazilian Internal Review Board as an anonymous unlinked study. This is a cross-sectional study included 267 pa-
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
tients that were failing HAART. These patients were randomly selected in AIDS clinics from five different Brazilian States. Therapeutic failure was defined as the persistence of viral load counts higher than 30 000 copies/ml after the continuous use of three or more ARV drugs, including at least one protease inhibitors (PI), for a period longer than 3 months. The starting material was viral RNA previously extracted for viral load measurement. After selecting the patients, the clinical and laboratory data (drug regimen, CD4 counts, and virus loads) were collected and patient identifying information removed from samples tubes. Plasma viral RNAs were isolated using a Organon Teknica Extraction Kit according to the manufacturer’s protocol, and stored at − 70 °C. Two different HIV-1 genomic regions were targeted for PCR amplification: pro and pol RT (palm finger region). Two-stage nested PCR amplification was performed as previously described (Tanuri et al., 1999b; Stuyver et al., 1997). Mutations related to RT drug resistance (L41M, T69D/ N and T215Y/F, L74V, and M184V, associated with AZT, ddI/ddC, and 3TC resistance, respectively) were analyzed using the line probe assay (LIPA) (Stuyver et al., 1997) using biotinilated PCR product following the manufacturer’s protocol. HIV-1 pro gene substitutions at positions 82 and/or 90 (associated with Indinavir, Ritonavir, Nelfinavir, and Saquinavir resistance) were tested using a restriction fragment length polymorphisms (RFLP) analysis with HincII digestion as previously described (Vasudevachari et al., 1996). Discrete variables are described in absolute counts and percents. Continuous data are described by the median and interquartile range. The distribution of categorical variables were compared with the use of the 2-test or the Fisher’s exact test. The Kruskal– Wallis test was used to compare the distribution of the viral load. Statistical analyses were performed with SPSS 9.0 for Windows (SPSS Inc.)
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32%), Sa˜ o Paulo (n =45; 17%), Santa Catarina (n= 67; 25%), and Espirito Santo (n= 24; 9%), and Brasilia (n= 43; 7%). When the patients drug regimen were analyzed, AZT and 3TC were the NRTIs more utilized, and Indinavir the main PI (Fig. 1). The main drug combination found in the treatment of patients engaged in this study were AZT +3TC + IDV (n = 56; 21%) followed by D4T +3TC + IDV (n = 32; 12%) and AZT+ DDI + IDV (n = 29; 11%). The genetic analyses of the pro and RT gene of the 267 patients in the study are shown in Table 1. There were 41 of patients (15.3%) with no mutations in either genomic region. In the remaining 226 patients (84.7%), resistance mutations were detected. Patients were divided into three groups according to the viral genetic patterns. In eighteen patients (6.7%), we observed wild type virus sequence in the RT gene and one or more mutations in the pro gene. The presence of mutations in the RT gene and wild type virus sequences in pro were found in 109 patients (40.8%). In the remaining 99 (37.2%), there were viral genomes carrying mutations in both regions. The proportion of patients with CD4 counts B 50 cells/mm3 was significantly associated with the distribution of resistance mutations (Table 1, PB 0.002). The groups that included patients with resistance mutations in the protease gene (with or without
3. Results Two hundred and sixty seven patients were selected in the states of Rio de Janeiro (n =86;
Fig. 1. Histogram showing the ARV drug used in the occasion of the patients were included in the study.
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A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
Table 1 CD4 counts and viral loads according to the presence of resistance mutations in RT and protease (pro) genes of HIV-1 Groups
Viral load (log) median (interquartile range)
CD4B50 cells/mm3, n (%)a
Wild type (n =41; 15.3%) Mutant RT (n = 109; 40.8%) Mutant pro (n = 18; 6.7%) Mutant pro and RT (n=99; 37.2%) Total (n =267; 100%)
5.32 (5.07–5.88)
5 (13)
5.2 (4.99–5.5)
16 (17)
5.4 (5.08–5.74)
7 (47)
5.38 (5.1–5.6)
28 (35)
P=0.078
P=0.002
RT, reverse transcriptase; pro, protease. a CD4 counts calculation based on 232 patients.
concomitant mutations in the RT gene) tended to have higher proportions of patients with CD4 counts below 50 cell/mm3. However, we could not find significant association between the variation in the viral load and these four groups of patients. The distribution of mutations in the RT and pro genes is depicted in Fig. 2. The main mutation found was M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). The main mutation pattern in the RT gene was the association of four mutations (M184V+ T69D/N + T215Y/F + M41L, n = 49, 18.4%). In the pro gene, the main mutation found was L90M (26%) followed by the dual substitution in L90M and V82A (6%). The mutation V82A alone was found in only 2.5% of the analyzed patients. In another analysis, the distribution of distinct viral genotypes was segregated by different Brazilian states. The frequence of wild type isolates were significantly associated with the geographic region where the samples were collected (P B 0.001; Fig. 3).
4. Discussion This is the first large molecular epidemiological survey in patients failing HAART therapy in Brazil. Our findings point out to a high prevalence of mutations related to drug resistance in RT
and/or pro genes in patients experiencing therapeutical failure defined by an elevated virus load (\30 000 copies/ml) . Resistance mutations in the RT and /or pro genes of HIV were detected in 85% of the patients that were not responding to HAART with the methods employed in this study. In another study, similar genetic analysis was performed in a cohort of 41 patients showing undetectable VL under ARV treatment in Rio de Janeiro (data not shown). In contrast with the results of this study, wild type was found in most patients (75%) in that study. Although our genetic screening methodology only analyzes few sorts of drug resistance mutation in RT (n= 5) and pro (n=2) they are primary drug resistant mutations and clearly could give a good picture about the level of genetic. However, the percentage of patients with mutated genomes could be higher than we observed. On the other hand, these methods are suitable for molecular epidemiological surveys due to their low cost and simplicity. AZT and 3TC were the NRTIs more used, and Indinavir the main PI as well the drug combination utilized in each of the patients. Early virologic failure of indinavir-AZT-3TC or amprenavir-AZT-3TC is associated initially with M184V substitution (Havlir et al., 2000). This fact explains the finding of mutation in the RT gene in a high proportion (40.8%) of isolates studied. We could not rule out the possibility that the presence of a higher number of individuals presenting genetic mutations on the RT gene found in our study could be associated with either a longer exposition to these drugs. Alternatively, this could be associated to a limited identification of mutations in the pro gene with the molecular screening method used in this study. We observed a significant variation in the proportion of wild type virus according to the geographic origin of the studied sample. This difference could be explained by the fact that the ARV drugs were first made available in Sao Paulo and Brasilia and probably the patients studied in these sites were exposed to ARV drugs longer than the other sites, driving the selection of resistant strains. Other explanation could be the different distribution of HIV-1 subtype in Brazil. The
Fig. 2. Histogram showing the frequency of the different resistance mutations of RT (substitutions analyzed by LIPA®: M41L, T69D/N, L74V, M184V, and T215Y/F), and protease (substitutions analyzed by RFLP: V82A, L90M, and V82A/L90M) genes found in the 267 patients analyzed.
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46 43
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A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
Brazilian epidemic is characterized by multiple HIV-1 group M subtypes, primarily subtype B and the less prevalent subtype F, but also subtypes C and D (Morgado et al., 1994; Sabino et al., 1996; Tanuri et al., 1999a). Subtype F is the most prevalent non-B variant and represents approximately 18% of HIV-1 isolates from southeastern Brazilian cities. An increasing prevalence of subtype C infections has also been recognized in Brazilian southern-most states (Janini et al., 1998). In addition, the presence of recombinants, potential mosaics and dual infections has been documented (Gomez-Cano et al., 1998; Tanuri et al., 1999b; Brindeiro et al., 1999; Ramos et al., 1999; Pieniazek et al., 1995). The possibility that HIV-1 subtypes may differ in regards to the susceptibility to ARV drugs can not be excluded at this time. Thus, the selective pressure of ARV drugs to increase the incidence of genotypes with resistance mutations may differ among HIV-1
subtypes. The different distribution of non-B subtype isolates among the five geographic regions studied could also impact in the performance of the genetic screening test utilized (LIPA®, and pro RFLP). In fact, the performance of LIPA-RT® assay using non-B subtype isolates is less sensitive and present lack of hybridization in some of probes included in the strips (Brindeiro et al., 1999). On the other hand, the presence of both HincII restriction sites in the Brazilian main nonB drug-naive isolates such as C, and F is universal, showing a genetic stability in this genomic region (data not shown). Then, the lost of these sites is well correlated to mutations in protease 82th and 90th positions. Alternatively, the geographic variation in the proportion of wild type virus in patients failing HAART could be associated with regional variations in adherence to therapy. Interestingly, the region with the highest proportion of wild type viruses (Santa Catarina
Fig. 3. Histogram showing the comparison of the prevalence of wild type isolates among patients from the five studied areas. SC, Santa Catarina; ES, Espı´rito Santo; RJ, Rio de Janeiro; SP, Sa˜ o Paulo; DF, Distrito Federal (Brasilia).
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
State) is also the one with the highest proportion of transmission of HIV in association with intravenous drug abuse, a condition associated with low adherence to therapy. This study was not designed to address the ARV compliance profile of the studied patients. However, we speculate that the finding of elevated levels of viral load in the absence of mutations related to drug resistance in 15.3% of patients studied might be due to a lack of compliance and therefore a low selective pressure. In summary, this study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance. The selection of drug-resistant variants will be an important public health issue and could put at risk the efforts in controlling AIDS mortality by ARV therapy. Furthermore these drug-resistant variants can be transmitted (Little et al., 1999; Boden et al., 1999) and start an epidemic of drug resistant HIV-1 in countries where HAART therapy is widespread.
References Boden D, Hurley A, Zhang L, Cao Y, Guo Y, Jones E, et al. HIV-1 drug resistance in newly infected individuals. JAMA 1999;282(12):1135 –41. Brindeiro R, Vanderborght B, Caride E, Correa L, Oravec RM, Berro O, et al. Sequence diversity of the reverse transcriptase of human immunodeficiency virus type 1 from untreated Brazilian individuals. Antimicrob Agents Chemother 1999;43(7):1674 –80. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV1 in patients on highly active antiretroviral therapy. Science 1997;278(14):1295 –300. Gomez-Cano M, Rubio A, Puig T, Perez-Olmeda M, Ruiz L, Soriano V, et al. Prevalence of genotypic resistance to nucleoside analogues in antiretroviral-naive and antiretroviral-experienced HIV-infected patients in Spain. AIDS 1998;12(9):1015 – 20. Havlir DV, Hellmann NS, Petropoulos CJ, Whitcomb JM, Collier AC, Hirsch MS, et al. Drug susceptibility in HIV infection after viral rebound in patient receiving indinavir-containing regimens. JAMA 2000;283(2):229 –34. Hu DJ, Dondero TJ, Rayfield MA, George JR, Schochetman G, Jaffe HW, et al. The emerging genetic diversity of HIV. The importance of global surveillance for diagnostics, research, and prevention. JAMA 1996;275:210 –6.
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Janini LM, Tanuri A, Schechter M, Peralta JM, Vicente AC, Dela Torre N, et al. Horizontal and vertical transmission of human immunodeficiency virus type 1 dual infections caused by viruses of subtypes B and C. J Infect Dis 1998;177:227 – 31. Ledergerber B, Egger M, Opravil M, Telenti A, Hirschel B, Battegay M, et al. Clinical progression and virological failure on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study. Lancet 1999;353:863 – 8. Little SJ, Daar ES, D’Aquila RT, Keiser PH, Connick E, Whitcomb JM, et al. Reduced antiretroviral drug susceptibility among patients with primary HIV infection. JAMA 1999;282(12):1142 – 9. Morgado MG, Sabino EC, Shpaer EG, Bongertz V, Brigido L, Guimaraes MD, et al. V3 region polymorphism in HIV-1 from Brazil: prevalence of subtype B strains divergent from North American/European prototype and detection of subtype F. AIDS Res Hum Retroviruses 1994;10:569 – 76. Pieniazek D, Janini LM, Ramos A, Tanuri A, Schechter M, Peralta JM, et al. HIV-1 patients may harbor viruses of different phylogenetic subtypes: implications for the evolution of the HIV/AIDS pandemic. Emerg Infect Dis 1995;1:86 – 8. Ramos A, Tanuri A, Schechter M, Rayfield MA, Hu DJ, Cabral MC, et al. Dual and Recombinant Infections: An Integral Part of the HIV-1 Epidemic in Brazil. Emerg Infect Dis 1999;5(1):65 – 74. Sabino EC, Diaz RS, Brigido LF, Learn GH, Mullins JI, Reingold AL, et al. Distribution of HIV-1 subtypes seen in an AIDS clinic in Sao Paulo City, Brazil. AIDS 1996;10:1579 – 84. Shafer RW, Winters MA, Palmer S, Merigan TC. Multiple concurrent reverse transcriptase and protease mutations multidrug resistance of HIV-1 isolates from heavily treated patients. Ann Intern Med 1998;128(11):906 – 11. Stuyver L, Wyseur A, Rombout A, Louwagie J, Scarcez T, Verhofstede C, et al. Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. Antimicrob Agents Chemother 1997;41(2):284 – 91. Tanuri A, Swanson P, Devare S, Berro OJ, Savedra A, Costa LJ, et al. HIV-1 subtypes among blood donors from Rio de Janeiro, Brazil. J Acquir Immune Defic Syndr Hum Retrovirol 1999a;20(1):60 – 6. Tanuri A, Vicente AC, Otsuki K, Ramos CA, Ferreira OC Jr., Schechter M, et al. Genetic variation and susceptibilities to protease inhibitors among subtypes B and F isolates in Brazil. Antimicrob Agents Chemother 1999b;43(2):253 – 8. Vasudevachari MB, Zhang YM, Imamichi H, Imamichi T, Falloon J, Salzman NP. Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening pro-
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Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART Amilcar Tanuri a,*, Elena Caridea a, Maria C. Dantas b, Marisa G. Morgado c, Daise L.C. Mello c, Sandra Borges j, Marisa Tavares d, Selma B. Ferreira d, Guilherme Santoro-Lopes d, Claudia R.F. Martins f, Andre´ L.C. Esteves f, Ricardo S. Diaz h, Sandra M.S. Andreo h, Luiz A.P. Ferreira k, Rodrigo Rodrigues e, Tania Reuter e, Ana M.S. Cavalcanti g, Suelene M. de Oliveira g, Heraclito B. de Barbosa i, Paulo R. Teixeira b, Pedro N. Chequer b a
Departamento de Gene´tica, Laborato´rio de Virologia Molecular, Instituto de Biologia, Uni6ersidade Federal do Rio de Janeiro, CCS, Bloco A, sala 121, 2o andar, Rio de Janeiro, RJ, Cep: 21944 -970,. Brazil b Programa Brasileiro de AIDS/STD, Brazilian Ministry of Health, Brasilia, Brazil c Laborato´rio de Imunologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil d Laborato´rio Petrobras, Hospital Uni6ersita´rio Clementino Fraga Filho, UFRJ, Rio de Janeiro, Brazil e Nucleo de Doenc¸as Infeciosas, Uni6ersidade Federal do Espirito Santos, Espirito Santos, Brazil f Laborato´rio de Virologia, Uni6ersidade de Brası´lia, Brasilia, Brazil g Laborato´rio Central de Sau´de Pu´blica, Recife, Pernambuco, Brazil h Laborato´rio de Retro6irologia, Uni6ersidade Federal do Estado de Sa˜o Paulo, Sao Paulo, Brazil i Departamento de Epidemiologia, Uni6ersidade de Sa˜o Paulo, Sao Paulo, Brazil j Laborato´rio Central de Sau´de Pu´blica, Florianopolis, Santa Catarina, Brazil k Uni6ersidade Federal de Santa Catarina, Florianopolis, Santa Catarina, Brazil Received 11 July 2001; accepted 20 November 2001
Abstract Background: Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. Objecti6e: In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. Study Design: Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations
* Corresponding author. Tel.: + 55-21-280-8043; fax: + 55-21-280-0994 E-mail address: [email protected] (A. Tanuri). 1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 1 ) 0 0 2 4 9 - 9
40
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
related to drug resistance in RT gene was analyzed using a line probe assay (LIPA®) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. Results: There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. Conclusions: This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HIV-1; Protease; Reverse transcriptase; Phenotyping; Genotyping
1. Introduction Recent development of a new generation of drugs against HIV has led to marked changes in antiretroviral (ARV) therapy and treatment of HIV infection. ARV drug combinations including two nucleoside reverse transcriptase (RT) inhibitors and/or non-nucleoside RT inhibitors can reduce the viral load in plasma to undetectable levels and provide durable clinical benefits (Vella et al., 1996; Zhang et al., 1999). However, complete suppression of HIV-1 replication is rarely achieved with mono or dual therapy, which have a transient effect favoring the development of ARV resistance and cross resistance. For this reason, current guidelines for ARV therapy recommend at least triple-drug combination, the socalled highly active antiretroviral therapy (HAART). However, development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV (Shafer et al., 1998; Ledergerber et al., 1999). Continuous in vivo HIV replication and the error prone nature of the viral RT are the driving forces for generation of drug resistance (Hu et al., 1996). Moreover, recent studies identified replication-competent HIV-1 reservoirs in patients under HAART, raising a serious concern as to whether antiviral therapy can eradicate HIV-1 (Finzi et al., 1997; Zhang et al., 1999). The appearance of viruses resistant to ARV drugs is one of the reasons for therapeutic failure, characterized broadly as inadequate viral suppression, unsatisfactory increase in CD4+ T-cell counts, or clinical progression. This failure may be associated with other factors such as patient adherence, pharmacological factors or emergence
of resistant viruses. Moreover, there is increasing concern due to their further transmission of these resistant strains (Gomez-Cano et al., 1998; Tanuri et al., 1999b; Brindeiro et al., 1999). The Brazilian Ministry of Health (MOH) has been sponsoring a policy of universal access to ARV drugs for AIDS patients since 1996. By January 2001, Brazil had about 100 000 patients using these drugs in the Public Health System. The Brazilian MOH has adopted periodically reviewed guidelines to ARV treatment, in order to facilitate adequate and uniform prescription all over the country, as well as guarantee a more rational and logistical approach of the use of these drugs in the Public Health System. Moreover, the Brazilian MOH implemented a network of 70 laboratories distributed throughout the country to quantify viral load and measure CD4 + T-cell count in every HIV-infected individual treated in a public institution, allowing better monitoring of these individuals. The percentage of these patients experiencing virological failure related to drug-resistant strains is largely unknown. These patients could be a source of transmission of these variants to the recently infected individuals, starting a new epidemic wave. This work describes the ARV resistance profile in patients failing HAART in Brazil, in an attempt to correlate the HIV therapeutic failure with RT and protease resistance mutations.
2. Materials and methods This study was approved by Brazilian Internal Review Board as an anonymous unlinked study. This is a cross-sectional study included 267 pa-
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46
tients that were failing HAART. These patients were randomly selected in AIDS clinics from five different Brazilian States. Therapeutic failure was defined as the persistence of viral load counts higher than 30 000 copies/ml after the continuous use of three or more ARV drugs, including at least one protease inhibitors (PI), for a period longer than 3 months. The starting material was viral RNA previously extracted for viral load measurement. After selecting the patients, the clinical and laboratory data (drug regimen, CD4 counts, and virus loads) were collected and patient identifying information removed from samples tubes. Plasma viral RNAs were isolated using a Organon Teknica Extraction Kit according to the manufacturer’s protocol, and stored at − 70 °C. Two different HIV-1 genomic regions were targeted for PCR amplification: pro and pol RT (palm finger region). Two-stage nested PCR amplification was performed as previously described (Tanuri et al., 1999b; Stuyver et al., 1997). Mutations related to RT drug resistance (L41M, T69D/ N and T215Y/F, L74V, and M184V, associated with AZT, ddI/ddC, and 3TC resistance, respectively) were analyzed using the line probe assay (LIPA) (Stuyver et al., 1997) using biotinilated PCR product following the manufacturer’s protocol. HIV-1 pro gene substitutions at positions 82 and/or 90 (associated with Indinavir, Ritonavir, Nelfinavir, and Saquinavir resistance) were tested using a restriction fragment length polymorphisms (RFLP) analysis with HincII digestion as previously described (Vasudevachari et al., 1996). Discrete variables are described in absolute counts and percents. Continuous data are described by the median and interquartile range. The distribution of categorical variables were compared with the use of the 2-test or the Fisher’s exact test. The Kruskal– Wallis test was used to compare the distribution of the viral load. Statistical analyses were performed with SPSS 9.0 for Windows (SPSS Inc.)
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32%), Sa˜ o Paulo (n =45; 17%), Santa Catarina (n= 67; 25%), and Espirito Santo (n= 24; 9%), and Brasilia (n= 43; 7%). When the patients drug regimen were analyzed, AZT and 3TC were the NRTIs more utilized, and Indinavir the main PI (Fig. 1). The main drug combination found in the treatment of patients engaged in this study were AZT +3TC + IDV (n = 56; 21%) followed by D4T +3TC + IDV (n = 32; 12%) and AZT+ DDI + IDV (n = 29; 11%). The genetic analyses of the pro and RT gene of the 267 patients in the study are shown in Table 1. There were 41 of patients (15.3%) with no mutations in either genomic region. In the remaining 226 patients (84.7%), resistance mutations were detected. Patients were divided into three groups according to the viral genetic patterns. In eighteen patients (6.7%), we observed wild type virus sequence in the RT gene and one or more mutations in the pro gene. The presence of mutations in the RT gene and wild type virus sequences in pro were found in 109 patients (40.8%). In the remaining 99 (37.2%), there were viral genomes carrying mutations in both regions. The proportion of patients with CD4 counts B 50 cells/mm3 was significantly associated with the distribution of resistance mutations (Table 1, PB 0.002). The groups that included patients with resistance mutations in the protease gene (with or without
3. Results Two hundred and sixty seven patients were selected in the states of Rio de Janeiro (n =86;
Fig. 1. Histogram showing the ARV drug used in the occasion of the patients were included in the study.
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Table 1 CD4 counts and viral loads according to the presence of resistance mutations in RT and protease (pro) genes of HIV-1 Groups
Viral load (log) median (interquartile range)
CD4B50 cells/mm3, n (%)a
Wild type (n =41; 15.3%) Mutant RT (n = 109; 40.8%) Mutant pro (n = 18; 6.7%) Mutant pro and RT (n=99; 37.2%) Total (n =267; 100%)
5.32 (5.07–5.88)
5 (13)
5.2 (4.99–5.5)
16 (17)
5.4 (5.08–5.74)
7 (47)
5.38 (5.1–5.6)
28 (35)
P=0.078
P=0.002
RT, reverse transcriptase; pro, protease. a CD4 counts calculation based on 232 patients.
concomitant mutations in the RT gene) tended to have higher proportions of patients with CD4 counts below 50 cell/mm3. However, we could not find significant association between the variation in the viral load and these four groups of patients. The distribution of mutations in the RT and pro genes is depicted in Fig. 2. The main mutation found was M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). The main mutation pattern in the RT gene was the association of four mutations (M184V+ T69D/N + T215Y/F + M41L, n = 49, 18.4%). In the pro gene, the main mutation found was L90M (26%) followed by the dual substitution in L90M and V82A (6%). The mutation V82A alone was found in only 2.5% of the analyzed patients. In another analysis, the distribution of distinct viral genotypes was segregated by different Brazilian states. The frequence of wild type isolates were significantly associated with the geographic region where the samples were collected (P B 0.001; Fig. 3).
4. Discussion This is the first large molecular epidemiological survey in patients failing HAART therapy in Brazil. Our findings point out to a high prevalence of mutations related to drug resistance in RT
and/or pro genes in patients experiencing therapeutical failure defined by an elevated virus load (\30 000 copies/ml) . Resistance mutations in the RT and /or pro genes of HIV were detected in 85% of the patients that were not responding to HAART with the methods employed in this study. In another study, similar genetic analysis was performed in a cohort of 41 patients showing undetectable VL under ARV treatment in Rio de Janeiro (data not shown). In contrast with the results of this study, wild type was found in most patients (75%) in that study. Although our genetic screening methodology only analyzes few sorts of drug resistance mutation in RT (n= 5) and pro (n=2) they are primary drug resistant mutations and clearly could give a good picture about the level of genetic. However, the percentage of patients with mutated genomes could be higher than we observed. On the other hand, these methods are suitable for molecular epidemiological surveys due to their low cost and simplicity. AZT and 3TC were the NRTIs more used, and Indinavir the main PI as well the drug combination utilized in each of the patients. Early virologic failure of indinavir-AZT-3TC or amprenavir-AZT-3TC is associated initially with M184V substitution (Havlir et al., 2000). This fact explains the finding of mutation in the RT gene in a high proportion (40.8%) of isolates studied. We could not rule out the possibility that the presence of a higher number of individuals presenting genetic mutations on the RT gene found in our study could be associated with either a longer exposition to these drugs. Alternatively, this could be associated to a limited identification of mutations in the pro gene with the molecular screening method used in this study. We observed a significant variation in the proportion of wild type virus according to the geographic origin of the studied sample. This difference could be explained by the fact that the ARV drugs were first made available in Sao Paulo and Brasilia and probably the patients studied in these sites were exposed to ARV drugs longer than the other sites, driving the selection of resistant strains. Other explanation could be the different distribution of HIV-1 subtype in Brazil. The
Fig. 2. Histogram showing the frequency of the different resistance mutations of RT (substitutions analyzed by LIPA®: M41L, T69D/N, L74V, M184V, and T215Y/F), and protease (substitutions analyzed by RFLP: V82A, L90M, and V82A/L90M) genes found in the 267 patients analyzed.
A. Tanuri et al. / Journal of Clinical Virology 25 (2002) 39–46 43
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Brazilian epidemic is characterized by multiple HIV-1 group M subtypes, primarily subtype B and the less prevalent subtype F, but also subtypes C and D (Morgado et al., 1994; Sabino et al., 1996; Tanuri et al., 1999a). Subtype F is the most prevalent non-B variant and represents approximately 18% of HIV-1 isolates from southeastern Brazilian cities. An increasing prevalence of subtype C infections has also been recognized in Brazilian southern-most states (Janini et al., 1998). In addition, the presence of recombinants, potential mosaics and dual infections has been documented (Gomez-Cano et al., 1998; Tanuri et al., 1999b; Brindeiro et al., 1999; Ramos et al., 1999; Pieniazek et al., 1995). The possibility that HIV-1 subtypes may differ in regards to the susceptibility to ARV drugs can not be excluded at this time. Thus, the selective pressure of ARV drugs to increase the incidence of genotypes with resistance mutations may differ among HIV-1
subtypes. The different distribution of non-B subtype isolates among the five geographic regions studied could also impact in the performance of the genetic screening test utilized (LIPA®, and pro RFLP). In fact, the performance of LIPA-RT® assay using non-B subtype isolates is less sensitive and present lack of hybridization in some of probes included in the strips (Brindeiro et al., 1999). On the other hand, the presence of both HincII restriction sites in the Brazilian main nonB drug-naive isolates such as C, and F is universal, showing a genetic stability in this genomic region (data not shown). Then, the lost of these sites is well correlated to mutations in protease 82th and 90th positions. Alternatively, the geographic variation in the proportion of wild type virus in patients failing HAART could be associated with regional variations in adherence to therapy. Interestingly, the region with the highest proportion of wild type viruses (Santa Catarina
Fig. 3. Histogram showing the comparison of the prevalence of wild type isolates among patients from the five studied areas. SC, Santa Catarina; ES, Espı´rito Santo; RJ, Rio de Janeiro; SP, Sa˜ o Paulo; DF, Distrito Federal (Brasilia).
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State) is also the one with the highest proportion of transmission of HIV in association with intravenous drug abuse, a condition associated with low adherence to therapy. This study was not designed to address the ARV compliance profile of the studied patients. However, we speculate that the finding of elevated levels of viral load in the absence of mutations related to drug resistance in 15.3% of patients studied might be due to a lack of compliance and therefore a low selective pressure. In summary, this study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance. The selection of drug-resistant variants will be an important public health issue and could put at risk the efforts in controlling AIDS mortality by ARV therapy. Furthermore these drug-resistant variants can be transmitted (Little et al., 1999; Boden et al., 1999) and start an epidemic of drug resistant HIV-1 in countries where HAART therapy is widespread.
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