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Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-05T19:55:42Z No. of bitstreams: 2 Dissertação- Luciana Oliveira Bento - 2016.pdf: 3533613 bytes, checksum: f67fe73c6af12700e4cba39192ea7c95 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-08T14:23:27Z (GMT) No. of bitstreams: 2 Dissertação- Luciana Oliveira Bento - 2016.pdf: 3533613 bytes, checksum: f67fe73c6af12700e4cba39192ea7c95 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-08T14:23:27Z (GMT). No. of bitstreams: 2 Dissertação- Luciana Oliveira Bento - 2016.pdf: 3533613 bytes, checksum: f67fe73c6af12700e4cba39192ea7c95 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The prevalence of isolates of HIV-1 with resistance mutations to antiretroviral drugs should be monitored continuously and in different population groups from geographical regions Brazilian, since Brazil offers universal access to treatment for all people living with HIV and AIDS. Because of the scarcity of related studies in cities of the interior of Brazil, this study aimed to identify the HIV-1 genetic diversity and to evaluate the profile and the prevalence of HIV-1 isolates with mutations in non-antiretroviral (ARV) exposed patients attended at the Specialized Service in STD/AIDS of the Jataí city, southwestern of Goiás state. From January 2015 to January 2016, 57 patients not exposed to ARVs were recruited and whole blood samples were collected. The protease (PR) and about 2/3 of the reverse transcriptase (RT) regions were amplified in 46 samples by "nested"-PCR and sequenced. Resistance mutations to ARVs were determined by Calibrated Population Resistance Tool from Stanford University and HIV-1 subtypes were identified by REGA and phylogenetic inference. In this study, the prevalence of HIV-1 resistant was more frequent among young male population, heterosexual, especially in the reproductive age group and brown race. Among 46 HIV-1 isolates sequenced, 5 had primary resistance mutations to ARVs, giving a prevalence of 10.9%. The mutations were detected both non-nucleoside RT inhibitors-NNRTIs (K103N, E138K/A and V179E) and for PR inhibitors-IP (M46L and T74S). As the K103N mutation confers resistance to high profile ARV composing the first line of treatment (EFV), it was introduced into the second line with IP for this patient. The E138K mutation confers resistance to an ARV not used in Brazil (RPV), allowing the introduction of the first line of treatment with the fixed-dose combination (formulation 3 in 1). Two isolates of HIV-1 were IP resistance mutations (M46L and T74S) but not yet started therapy. In this case, the introduction of the treatment with the formulation 3 to 1 is possible, since the first line has no IP in its formulation. HIV-1 subtype B was the prevalent isolates and three recombinants involving subtypes B and F1 were observed. The subtype C and recombinant forms were first reported in Goiás southwestern region. The moderate prevalence of primary resistance of HIV-1 isolates among patients from southwestern Goiás state and co-circulation of “pure” HIV-1 subtypes and recombinant forms, it is evident the importance of monitoring of newly diagnosed patients to optimize initial therapy, improving clinical management and control of transmission of HIV-1. / A prevalência de isolados do HIV-1 com mutações de resistência aos antirretrovirais deve ser monitorada continuamente nas diferentes regiões geográficas e grupos populacionais brasileiros, visto que o Brasil disponibiliza acesso universal ao tratamento para todas as pessoas vivendo com HIV e aids. Devido à escassez de estudos relacionados em cidades do interior do Brasil, este trabalho teve como objetivo identificar a diversidade genética do HIV-1 e avaliar o perfil e a prevalência de isolados do HIV-1 com mutações, em pacientes não expostos aos antirretrovirais (ARVs) atendidos no Serviço de Atendimento Especializado em DST/aids do município de Jataí, no sudoeste goiano. De janeiro de 2015 a janeiro de 2016, foram recrutados 57 pacientes não expostos aos ARVs e amostras de sangue total foram coletadas. Os genes da protease (PR) e cerca de 2/3 da transcriptase reversa (TR) foram amplificados em 46 amostras pela “nested”-PCR e sequenciados. As mutações de resistência aos ARVs foram determinadas mediante a ferramenta Calibrated Population Resistance Tool da Universidade de Stanford e os subtipos do HIV-1 foram identificados pela análise por REGA e inferência filogenética. Neste estudo, a prevalência da resistência aos antirretrovirais foi mais frequente na população jovem não exposta do sexo masculino, heterossexual, na cor parda, e especialmente nas faixas etárias de 30 a 34 anos, e de 40 a 49 anos de idade. Entre os 46 isolados de HIV-1 sequenciados, 5 apresentaram mutações de resistência primária aos ARVs, conferindo uma prevalência de 10,9%. Foram detectadas mutações tanto para inibidores da TR não nucleosídicos-NNRTI (K103N, E138K/A e V179E) quanto para inibidores da PR-IP (M46L e T74S). Como a mutação K103N confere alto perfil de resistência ao ARV que compõe o esquema de primeira linha de tratamento (EFV), foi introduzida a segunda linha com IP (LPV/r) para este paciente. A mutação E138K confere resistência a um ARV ainda não utilizado no Brasil (RPV), o que permitiu a introdução da primeira linha de tratamento constituída pela dose fixa combinada com TDF+3TC+EFV (formulação 3 em 1). Dois isolados do HIV-1 apresentaram mutações de resistência (M46L e T74S) que conferem resistência ao IP (NFV), mas ainda não iniciaram a terapia. Nesse caso, a introdução do tratamento com a formulação 3 em 1 será possível, já que a primeira linha não tem o IP (NFV) em sua formulação. O subtipo B do HIV-1 foi o prevalente e três isolados recombinantes foram observados, envolvendo os subtipos B e F1. O subtipo C e as formas recombinantes foram relatados pela primeira vez na região do sudoeste goiano. Com a identificação de uma prevalência moderada de isolados de HIV-1 com resistência primária entre pacientes do sudoeste goiano e a co-circulação de subtipos “puros” e mosaicos do HIV-1, fica evidente a importância do monitoramento dos pacientes recém-diagnosticados para a otimização da terapia inicial, melhorando a conduta clínica e o controle da transmissão do HIV-1.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-02-24T20:04:38Z No. of bitstreams: 2 Dissertação - Regyane Ferreira Guimarães Dias - 2015.pdf: 6640888 bytes, checksum: 32d26ee8f08840eec10eaca06583b3b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-02T11:00:27Z (GMT) No. of bitstreams: 2 Dissertação - Regyane Ferreira Guimarães Dias - 2015.pdf: 6640888 bytes, checksum: 32d26ee8f08840eec10eaca06583b3b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-02T11:00:27Z (GMT). No. of bitstreams: 2 Dissertação - Regyane Ferreira Guimarães Dias - 2015.pdf: 6640888 bytes, checksum: 32d26ee8f08840eec10eaca06583b3b8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The current scenario of HIV infection epidemic in Brazil includes "interiorization", "heterosexualization" and the increase of the emergence of resistant strains to antiretroviral therapy (ART). The Jataí city is responsible for diagnostic and treatment of HIV/aids patients from municipalities from Southwest region of Goiás state, and has been among the cities with the highest HIV/AIDS rate detection in the state. The epidemiological profile of the patients infected with HIV from small and medium-sized cities is still unknown, as well as the prevalence of ARV resistance. This study goal to trace the clinical and epidemiological profile of HIV/AIDS patients attended at Jataí city between 2005-2015 and, among patients under ART at the year 2015, the resistance mutations and the HIV-1 subtypes were determined. From 539 HIV/AIDS patients attended at Jataí, most were male, with infection prevalence among the age group of 30-34 and 40-49 years. Among female patients, the infection occurred mainly among 19-24 and 40-49 years. The sexual unprotected exposure category to HIV-1 prevailed, being the heterosexual preference predominant. The majority of patients were symptomatic at the diagnosis, being the weight loss the predominant complaint. The pneumocystosis and toxoplasmosis were the most frequent opportunistic infections related to AIDS. The AIDS was the cause of death in most cases. Regarding patients on ART, there was significant difference between the CD4+ T cell counts median and the viral load median (on admission) and at final (current lab result). The genotyping test for the subtypes identification and ARV resistance mutations was performed among 26 samples of ART patients, wherein 21 samples were amplified. The HIV-1 subtype B prevailed (19/21) and 2/21 classified as F1 subtype. The secondary resistance prevalence was 52.4%. Most isolates with resistance (7/11) had mutations at more than one ARV class and four isolates had mutations for a single class. The most frequent mutation was M184V, followed by K103N and thymidine associated mutations (TAMs-D67N, M41L, L210W and T215Y). The data presented in this study contributes to the HIV-1 diversity mapping and ARV resistance surveillance among patients from the Southwest region of Goiás state and will certainly be useful for local epidemiological surveillance specific actions. / O atual cenário da epidemia da infecção pelo HIV no Brasil inclui a “interiorização”, “heterossexualização” e aumento da emergência de cepas resistentes aos esquemas de terapia antirretroviral (TARV). O município de Jataí é responsável pelo diagnóstico e tratamento dos pacientes portadores de HIV/aids de municípios da regional Sudoeste II de Goiás e já esteve entre as cidades com a maior taxa de detecção de casos de aids do estado. O perfil epidemiológico dos pacientes infectados pelo HIV de municípios de pequeno e médio porte é ainda pouco conhecido, assim como a prevalência de resistência aos ARVs. Este estudo teve como objetivo traçar o perfil clínico e epidemiológico dos pacientes HIV+/aids atendidos em Jataí entre os anos de 2005 a 2015 e, entre os pacientes em TARV no ano de 2015, identificar mutações de resistência aos ARVs e os subtipos do HIV-1. De 539 pacientes HIV+/aids atendidos em Jataí, a maioria era do sexo masculino, com predomínio da infecção na faixa etária de 30 a 34 e 40 a 49 anos. Entre pacientes do sexo feminino, a infecção ocorreu predominantemente entre 19 a 24 e 40 a 49 anos. A categoria da exposição ao HIV-1 sexual sem proteção prevaleceu, sendo dominante a preferência heterossexual. A maioria dos pacientes era sintomático ao diagnóstico, sendo a perda ponderal a queixa predominante. A pneumocistose e a neurotoxoplasmose foram as infecções oportunistas relacionadas à aids mais frequentes e a aids foi a causa básica do óbito na maioria dos casos. Entre os pacientes em TARV houve diferença significante entre as medianas das contagens de células T CD4+ e entre as medianas da quantificação da carga viral inicial (na admissão) e final (atual). O teste de genotipagem para a identificação dos subtipos e resistência aos ARVs foi realizado em 26 amostras de pacientes em TARV, sendo 21 amostras amplificadas. O subtipo B prevaleceu (19/21) e 2/21 foram classificadas como subtipo F1. A prevalência de resistência secundária foi de 52,4%. A maioria dos isolados com resistência (7/11) apresentou mutações a mais de uma classe de ARVs e 4 isolados para uma única classe. A mutação mais frequente foi a M184V, seguida da K103N e de mutações associadas à timidina (TAMs-D67N, M41L, L210W e T215Y). Os dados apresentados neste estudo contribuem para o mapeamento da diversidade do HIV-1 e da vigilância da resistência aos ARVs em pacientes do Sudoeste goiano e, certamente serão úteis para o planejamento de ações específicas de vigilância epidemiológica local.

Nous avons effectué ce travail afin d’évaluer l’impact d’une utilisation accrue des antirétroviraux (ARV) sur l’émergence de la résistance dans le cadre d’une cohorte de sujets infectés par le VIH-1, enrôlés au Mali pour recevoir la thérapie antirétrovirale. La première partie de ce travail a évalué la résistance primaire auprès de 101 sujets naïfs aux ARV. Cette étude a démontré que la majorité des sujets (71,3%) étaient infectés par le sous-type CRF02_AG. La prévalence de la résistance primaire était de 9,9%. Ce chiffre dépasse largement la moyenne de 5,5% observée dans les pays en développement et le seuil des 5% fixé par l’OMS dans le cadre de la surveillance de la résistance. Les mutations associées aux analogues de la thymidine ou « Thymidine-associated Mutations » (TAMs): M41L, D67N, L210W, T215A/Y, K219E liées à la résistance aux inhibiteurs nucléosidiques de la transcriptase inverse (INTI) ainsi que les mutations K103N, V108I, V179E et Y181C impliquées dans la résistance aux inhibiteurs non nucléosidiques de la transcriptase inverse (INNTI) étaient majoritairement observées. Ces mutations sont compatibles avec les régimes de traitement de première ligne utilisés au Mali, composés de stavudine/lamivudine/nevirapine. Nous n’avons pas trouvé de mutations majeures aux inhibiteurs de protéase (IP), probablement du fait que cette classe d’ARV est rarement utilisée au Mali. Cependant plusieurs polymorphismes au niveau du gène de la protéase, particulièrement L10I et L10V ont été observés à une fréquence très élevée (18,80%). Compte tenu de ces premiers résultats, une suite logique de ce travail était de savoir comment des souches de sous-type CRF02_AG évolueraient sous la pression de sélection des ARV. Nous avons abordé ces questions dans une étude de cohorte de 132 sujets infectés majoritairement avec le sous-type CRF02_AG débutant une thérapie de première ligne. Nos résultats suggèrent que la présence de mutation de résistance primaire pourrait avoir un effet sur l’efficacité du traitement. Par exemple, la présence d’une seule mutation INNTI avant traitement comme K103N ou V179E était suffisante pour mener à l’échec au traitement (charge virale supérieure à 400 copies/ml). Par ailleurs, nous avons effectué des expériences in vitro pour mieux évaluer l’impact du polymorphisme L10I/V chez le sous-type CRF02_AG. Il faut savoir que le rôle de ce polymorphisme reste incertain chez le sous-type CRF02_AG, car aucune étude in vitro n’avait été réalisée auparavant. Nos résultats indiquent chez le sous-type sauvage CRF02_AGwt_10L une légère augmentation de la concentration inhibitrice 50% (IC50) pour le darunavir, le lopinavir et le nelfinavir comparativement au sous-type de référence B HXB2_10L avec respectivement un « Fold Change » (FC) de 1,2, 1,3 et 1,5. Cette augmentation est plus importante pour le lopinavir avec un FC (1,3) très proche de son seuil biologique (1,6). Comparativement au type sauvage CRF02_AGwt_10L, nos deux mutants CRF02_AGL10I et CRF02_AGL10V ont démontré une légère augmentation d’IC50 pour l’indinavir (avec respectivement un FC de 1,3 et 1,2) et une diminution pour le lopinavir (avec respectivement un FC de 0,78 et 0,75). Toutes ces observations suggèrent que la mutation en position 10 pourrait avoir un impact chez le sous-type CRF02_AG. Toutefois, la signification clinique de ces observations doit être déterminée. En conclusion, nos résultats supportent d’une part la nécessité de renforcer la surveillance de la résistance aux ARV et d’autre part, il fournit des informations nécessaires à l’amélioration des stratégies thérapeutiques afin de prévenir les échecs aux traitements chez les sous-types non B, particulièrement le CRF02_AG. / We conducted this study to assess the impact of an increased use of antiretroviral (ARV) treatment on the emergence of resistance in a cohort of subjects infected with HIV-1 enrolled in Mali to receive antiretroviral therapy. The first part of this work evaluated the incidence of primary resistance with 101 ARV treatment naive subjects. We demonstrated that the majority of subjects (71.3%) were infected with subtype CRF02_AG. The prevalence of primary resistance was 9.9%. This rate exceeds the average of 5.5% observed in developing countries and the 5% threshold set by the WHO as part of the HIV drug resistance surveillance. The thymidine-associated mutations (TAMs) M41L, D67N, L210W, T215A/Y, K219E, which are associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs), and K103N, V108I, Y181C and V179E, which are involved in resistance to none nucleoside reverse transcriptase inhibitors (NNRTIs), were predominantly observed. These mutations are compatible with the first line regimens used in Mali, including stavudine, lamivudine, and nevirapine. We did not find major mutations associated with resistance to protease inhibitors (PI) probably because this class of drugs is rarely used in Mali. However, several polymorphisms in the protease gene such as L10I and L10V were observed at a very high frequency (18.80%). Given these initial results, a logical extension of this work was to evaluate how these strains evolve under the selective pressure of ARV treatment. We have addressed these issues in a cohort study of 132 subjects predominantly infected with the subtype CRF02_AG that started a first-line therapy. Our results suggest that the presence of primary resistance mutation could affect treatment efficacy. For example, the presence of a single NNRTI mutation K103N or V179E before treatment initiation was sufficient to lead to treatment failure (viral load > 400 copies/ml). Otherwise, the role of L10I/V polymorphism remains uncertain in the CRF02_AG subtype. Therefore, we performed in vitro experiments to assess the impact of this polymorphism in subtype CRF02_AG on resistance to PI. Our results indicate in wild type CRF02_AGwt_10L a slight increase of the half maximal inhibitory concentration (IC50) for darunavir, lopinavir and nelfinavir compared with subtype B reference HXB2_10L with a Fold Change (FC) 1.2, 1.3 and 1.5, respectively. This increase was the greatest for lopinavir with a FC (1.3) very close to his biological threshold (1.6). Compared to wild type CRF02_AGwt_10L, the two mutants CRF02_AGL10I and CRF02_AGL10V showed a slight increase of IC50 for indinavir (FC of 1.3 and 1.2, respectively) and lower for lopinavir (FC of 0.78 and 0.75, respectively). All these findings suggest that the mutation at position 10 may have an impact on resistance to PI in subtype CRF02_AG. However, the clinical significance of these observations remains to be determined. In conclusion, our results support the need for strengthening HIV drug resistance surveillance and provide information toward the improvement of therapeutic strategies to prevent treatment failure in non-B subtype, particularly the subtype CRF02_AG.

Nous avons effectué ce travail afin d’évaluer l’impact d’une utilisation accrue des antirétroviraux (ARV) sur l’émergence de la résistance dans le cadre d’une cohorte de sujets infectés par le VIH-1, enrôlés au Mali pour recevoir la thérapie antirétrovirale. La première partie de ce travail a évalué la résistance primaire auprès de 101 sujets naïfs aux ARV. Cette étude a démontré que la majorité des sujets (71,3%) étaient infectés par le sous-type CRF02_AG. La prévalence de la résistance primaire était de 9,9%. Ce chiffre dépasse largement la moyenne de 5,5% observée dans les pays en développement et le seuil des 5% fixé par l’OMS dans le cadre de la surveillance de la résistance. Les mutations associées aux analogues de la thymidine ou « Thymidine-associated Mutations » (TAMs): M41L, D67N, L210W, T215A/Y, K219E liées à la résistance aux inhibiteurs nucléosidiques de la transcriptase inverse (INTI) ainsi que les mutations K103N, V108I, V179E et Y181C impliquées dans la résistance aux inhibiteurs non nucléosidiques de la transcriptase inverse (INNTI) étaient majoritairement observées. Ces mutations sont compatibles avec les régimes de traitement de première ligne utilisés au Mali, composés de stavudine/lamivudine/nevirapine. Nous n’avons pas trouvé de mutations majeures aux inhibiteurs de protéase (IP), probablement du fait que cette classe d’ARV est rarement utilisée au Mali. Cependant plusieurs polymorphismes au niveau du gène de la protéase, particulièrement L10I et L10V ont été observés à une fréquence très élevée (18,80%). Compte tenu de ces premiers résultats, une suite logique de ce travail était de savoir comment des souches de sous-type CRF02_AG évolueraient sous la pression de sélection des ARV. Nous avons abordé ces questions dans une étude de cohorte de 132 sujets infectés majoritairement avec le sous-type CRF02_AG débutant une thérapie de première ligne. Nos résultats suggèrent que la présence de mutation de résistance primaire pourrait avoir un effet sur l’efficacité du traitement. Par exemple, la présence d’une seule mutation INNTI avant traitement comme K103N ou V179E était suffisante pour mener à l’échec au traitement (charge virale supérieure à 400 copies/ml). Par ailleurs, nous avons effectué des expériences in vitro pour mieux évaluer l’impact du polymorphisme L10I/V chez le sous-type CRF02_AG. Il faut savoir que le rôle de ce polymorphisme reste incertain chez le sous-type CRF02_AG, car aucune étude in vitro n’avait été réalisée auparavant. Nos résultats indiquent chez le sous-type sauvage CRF02_AGwt_10L une légère augmentation de la concentration inhibitrice 50% (IC50) pour le darunavir, le lopinavir et le nelfinavir comparativement au sous-type de référence B HXB2_10L avec respectivement un « Fold Change » (FC) de 1,2, 1,3 et 1,5. Cette augmentation est plus importante pour le lopinavir avec un FC (1,3) très proche de son seuil biologique (1,6). Comparativement au type sauvage CRF02_AGwt_10L, nos deux mutants CRF02_AGL10I et CRF02_AGL10V ont démontré une légère augmentation d’IC50 pour l’indinavir (avec respectivement un FC de 1,3 et 1,2) et une diminution pour le lopinavir (avec respectivement un FC de 0,78 et 0,75). Toutes ces observations suggèrent que la mutation en position 10 pourrait avoir un impact chez le sous-type CRF02_AG. Toutefois, la signification clinique de ces observations doit être déterminée. En conclusion, nos résultats supportent d’une part la nécessité de renforcer la surveillance de la résistance aux ARV et d’autre part, il fournit des informations nécessaires à l’amélioration des stratégies thérapeutiques afin de prévenir les échecs aux traitements chez les sous-types non B, particulièrement le CRF02_AG. / We conducted this study to assess the impact of an increased use of antiretroviral (ARV) treatment on the emergence of resistance in a cohort of subjects infected with HIV-1 enrolled in Mali to receive antiretroviral therapy. The first part of this work evaluated the incidence of primary resistance with 101 ARV treatment naive subjects. We demonstrated that the majority of subjects (71.3%) were infected with subtype CRF02_AG. The prevalence of primary resistance was 9.9%. This rate exceeds the average of 5.5% observed in developing countries and the 5% threshold set by the WHO as part of the HIV drug resistance surveillance. The thymidine-associated mutations (TAMs) M41L, D67N, L210W, T215A/Y, K219E, which are associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs), and K103N, V108I, Y181C and V179E, which are involved in resistance to none nucleoside reverse transcriptase inhibitors (NNRTIs), were predominantly observed. These mutations are compatible with the first line regimens used in Mali, including stavudine, lamivudine, and nevirapine. We did not find major mutations associated with resistance to protease inhibitors (PI) probably because this class of drugs is rarely used in Mali. However, several polymorphisms in the protease gene such as L10I and L10V were observed at a very high frequency (18.80%). Given these initial results, a logical extension of this work was to evaluate how these strains evolve under the selective pressure of ARV treatment. We have addressed these issues in a cohort study of 132 subjects predominantly infected with the subtype CRF02_AG that started a first-line therapy. Our results suggest that the presence of primary resistance mutation could affect treatment efficacy. For example, the presence of a single NNRTI mutation K103N or V179E before treatment initiation was sufficient to lead to treatment failure (viral load > 400 copies/ml). Otherwise, the role of L10I/V polymorphism remains uncertain in the CRF02_AG subtype. Therefore, we performed in vitro experiments to assess the impact of this polymorphism in subtype CRF02_AG on resistance to PI. Our results indicate in wild type CRF02_AGwt_10L a slight increase of the half maximal inhibitory concentration (IC50) for darunavir, lopinavir and nelfinavir compared with subtype B reference HXB2_10L with a Fold Change (FC) 1.2, 1.3 and 1.5, respectively. This increase was the greatest for lopinavir with a FC (1.3) very close to his biological threshold (1.6). Compared to wild type CRF02_AGwt_10L, the two mutants CRF02_AGL10I and CRF02_AGL10V showed a slight increase of IC50 for indinavir (FC of 1.3 and 1.2, respectively) and lower for lopinavir (FC of 0.78 and 0.75, respectively). All these findings suggest that the mutation at position 10 may have an impact on resistance to PI in subtype CRF02_AG. However, the clinical significance of these observations remains to be determined. In conclusion, our results support the need for strengthening HIV drug resistance surveillance and provide information toward the improvement of therapeutic strategies to prevent treatment failure in non-B subtype, particularly the subtype CRF02_AG.