A l’ère de l’objectif cible « 90-90-90 » de l’ONUSIDA de réduction de la pandémie liée à HIV, il est important d’évaluer régulièrement la cascade de prise en charge des personnes vivant avec le virus HIV afin d’en vérifier l’avancement et d’identifier d’éventuels obstacles à sa réalisation. Pour cela nous avons étudié tout d’abord efficacité du traitement antirétroviral (TAR) dans le sang de personnes nouvellement dépistées séropositives à Bamako (Mali). Dans un deuxième travail nous avons évalué la faisabilité des tests de charge virale et de résistance génotypique aux antirétroviraux à partir de sang total séché sur un support de type buvard (DBS). La troisième partie de nos travaux était consacrée à des aspects plus physiopathologiques avec l’évaluation du traitement sur les réservoirs salivaires et génitaux (patients de Bamako) et sur le microbiote vaginal, ainsi que l’étude du profil de résistance des souches archivées dans l’ADN cellulaire de biopsies rectales. Nous avons tout d’abord observé un taux important de perdus de vue à un an dans la cohorte de Bamako (environ 45%). Nous avons également constaté un taux élevé de résistance primaire aux ARV à Bamako et au Tchad (>15%). De manière rassurante, le succès virologique au bout de 1 an chez les personnes traitées était d’environ 90% ce qui correspond à l’objectif de l’ONUSIDA (3ème 90) et un faible niveau de mutations acquises a été observé chez ces personnes adhérentes au traitement. Nous avons démontré l’efficacité du TAR sur le compartiment salivaire et constaté une compartimentation du virus au niveau cervico-vaginal chez certaines femmes traitées (27%) présentant une excrétion virale au niveau vaginal avec une charge virale plasmatique indétectable et/ou des séquences génétiques différentes sur le gène pol entre le virus isolé dans la muqueuse et celui provenant du sang. De plus, une dysbiose était observée avant la mise sous traitement, avec normalisation de la flore sous TAR efficace. En ce qui concerne le travail sur les biopsies rectales, des profils similaires ont été observés entre la souche plasmatique majoritaire au moment de la mise sous TAR et celle archivée dans le rectum 1 à 5 ans après traitement. En conclusion, nos travaux apportent des informations nouvelles sur le déroulement des différentes étapes de la prise en charge de l’infection par HIV dans les pays en développement : tout d’abord une faible adhésion au traitement ce qui peut constituer un obstacle majeur à la réalisation du plan 90/90/90 ; une forte prévalence de la résistance primaire qui plaident en faveur de l’accessibilité aux différentes classes d’antirétroviraux et de leur utilisation rationnelle, de l’utilisation généralisée en routine des tests de charge virale et du développement d’un réseau de surveillance de la résistance aux ARV dans les pays à ressources limitées ; des données d’efficacité de traitement sur les réservoirs muqueux mettant en évidence l’existence d’une dysbiose et d’une compartimentation du virus au niveau génital ce qui pose le problème du risque résiduel de transmission chez certaines personnes, même sous ARV. / Regularly assess to UNAIDS cascade 90-90-90 is important to check the progress and identify any obstacles to its implementation. For this we first studied efficacy of antiretroviral treatment (ART) in the blood of newly diagnosed HIV-positive in Bamako (Mali).In a second work we evaluated the feasibility of viral load and genotypic resistance tests from dried blood spot (DBS). The third part of our work is dedicated to pathophysiological aspects with evaluation of treatment on salivary and genital reservoirs (Bamako patients) and on the vaginal microbiota, as well as the study of the resistance profile of the strains archived in cellular DNA of rectal biopsies. We observed a high rate of lost to follow-up at one year in the Bamako cohort (45%). We also found a high rate of ART primary resistance in Bamako and Chad (> 15%). Reassuringly, the virological success after 1 year of treated follow was about 90% in these adherents. We also demonstrated the efficacy of ART in the salivary compartment and found a compartmentalization of the virus at the cervico-vaginal level in some women under ART. In addition, a dysbiosis was observed before ART, and a normal flora under effective ART. Similar profiles were observed on the main strain isolated in blood at the time of diagnosis and on the archived strain in the rectum after 1 to 5 years of ART.In conclusion, our work provides new information on the progress of the treatment stages of HIV infection in developing countries: low adherence to treatment which can constitute a major obstacle to achieve the plan 90/90/90; a high prevalence of primary resistance advocating accessibility and rational use of different classes of antiretrovirals drugs, widespread routine use of viral load tests and the development of ARVs resistance surveillance network in resource-limited countries; treatment efficacy data on mucosal reservoirs revealing the existence of genital dysbiosis and viral compartmentalization, which raises the problem of the residual risk of transmission in some people, even under ARVs.

Made available in DSpace on 2015-07-22T20:50:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-28. Added 1 bitstream(s) on 2015-08-11T03:25:28Z : No. of bitstreams: 1 Publico-00284.pdf: 1210351 bytes, checksum: 9468c9b00f4f6d4853d0e556a03096c4 (MD5) / Introdução: a falha ao HAART torna importante o desenvolvimento de novos fármacos que alvejam diferentes passos do ciclo de vida do HIV-1. O Enfuvirtide (T20) é um peptídeo sintético que mimetiza a região HR2 da gp41 do HIV-1, impedindo sua fusão e entrada na célula hospedeira. A presença de mutações de resistência primária ao T20 pode levar a falta de resposta virológica sustentada (RVS) em indivíduos em falha terapêutica ao HAART. Objetivos: genotipar a gp41 do HIV-1 de indivíduos considerados respondedores e não respondedores ao T20; verificar se a presença de mutações de resistência primária poderia interferir na RVS; correlacionar o status RVS com aspectos virais, imunológicos e tropismo. Metodologia: DNA genômico do baseline (antes do tratamento), 6 e 12 meses após o tratamento com T20, foi purificado utilizando QIAamp DNA Mini kit (Qiagen®, Valencia, Califórnia, USA). Todos pacientes receberam terapia otimizada mais T20 no inicio. 506 pb referentes as regiões HR1 e HR2 da gp41 foram amplificados por PCR “nested”. A PCR “nested” da região V3 para estudo do tropismo viral amplificou 654 pb. A PCR foi purificada utilizando Montage® PCR Centrifugal Filter Devises (Millipore®). As PCRs purificadas das regiões V3 e gp41 foram sequenciadas no ABI Prism 3130 Genetic Analyser (Applied Biosystems, Ca, USA) com o kit comercial BigDye® Terminator Cycle Sequencing versão 3.1 (Applied Biossystems, Foster City. Califórnia, USA). Iniciadores da segunda etapa da PCR “nested” foram utilizados para sequenciar. Resultados: sete indivíduos apresentavam um perfil de resposta ao T20 por 12 meses, 4 por pelo menos 6 meses e dois não responderam entre 6 e 12 meses. A média de idade foi de 44,92 ± 5,39, sendo 46,16% do sexo feminino e 53,84% do masculino. Dos 13 pacientes analisados, 12 pertencem ao subtipo B e 1 ao F1. Oito pacientes apresentaram o correceptor R5 e cinco o X4. Não foram encontradas mutações nas posições 36 a 45 da HR1 em 12 meses. Tivemos a N42S, que é responsável pela diminuição da susceptibilidade ao T20. De um total de 59 aminoácidos analisados na HR1, observamos 18,64% de trocas e na região HR2, 38,88%. Na HR1 no período de 8 meses ou mais em relação ao baseline, não encontramos modificações nas posições 36 a 45. De um total de 59 aminoácidos analisados, observamos 15,25% de trocas e na HR2, 36,11%. Verificamos a E137K sem a presença da N43D, que causou resistência. Verificamos a S138T como mutação de primaria com persistência de 12 meses sem a N43D e a falta de resposta ao tratamento. Na contagem de T CD4+, não encontramos diferença estatística entre as médias dos diferentes grupos (ANOVA – p=0,1). As cargas virais após 6 meses foram indetectáveis (<50 copias/mL) em 69,23% dos pacientes. Houve queda de carga viral do baseline para 6 meses (p=0.034), assim como para 6 meses e 8 meses ou mais. O uso do medicamento teve interferência significativa quando comparados o baseline e 6 meses (p=0.004) e entre o baseline e 12 meses (p=0.022), mas entre 6 e 12 meses a interferência não foi significativa, mostrando que após 6 meses ela permanece sustentada. Entre as variáveis, indivíduos respondedores e genótipo R5 houve alto valor de similaridade (76.98). Ocorreu agrupamento entre as duas amostras dos respondedores com genótipo X4 com as mutações 306, 311 e 320, que o caracterizam. Discussão: em relação ao tropismo, 38,47% dos pacientes que estudamos apresentaram correceptor X4 após alguns anos de infecção e múltiplos esquemas HAART. Este resultado mostra concordância com a literatura, que descreve uma taxa média de 50%. A N42S, observada em nosso estudo é relacionada com diminuição da susceptibilidade da droga. Apesar disso, somente um dos pacientes não respondeu ao tratamento, não obtendo ganho imunológico. Conclusão: o tropismo dos vírus HIV-1 na maioria dos indivíduos que responderam ao T20 está fortemente associado com o R5, que não causa progressão rápida. A presença da S138T foi suficiente para a falta de resposta ao T20, sem associação com a N43D. Mostramos evidente RVS e recuperação imunológica com o T20, mas que não foi suficiente para retirar estes indivíduos do risco de doenças oportunistas. Nosso estudo evidenciou a importância de iniciar o resgate quando a contagem de linfócitos T CD4+ está acima de 200 células/mm3. / Introduction: the failure to HAART becomes important to development of new drugs that target different steps of the life cycle of HIV-1. The Enfuvirtide (T20) is a synthetic peptide that mimics the HR2 region of gp41 of HIV-1, preventing its fusion and entry into the host cell. The presence of primary resistance mutations to T20 can lead to lack of sustained virologic response (SVR) in people in the HAART failure. Objectives: to genotype the gp41 of HIV-1 subjects considered responders and nonresponders to T20; verify the presence of primary resistance mutations could influence the SVR, SVR status correlate with aspects of viral, immunological and tropism. Methodology: genomic DNA from baseline (before treatment), 6 and 12 months after treatment with T20, was purified using QIAamp DNA Mini kit (Qiagen ®, Valencia, California, USA). All patients received optimal therapy more T20 at the beginning. 506 bp referring to the HR1 and HR2 regions of gp41 were amplified by PCR nested. PCR nested the V3 region to study viral tropism amplified 654 bp. The PCR was purified using Montage ® PCR Centrifugal Filter Devises (Millipore ®). The PCRs of purified V3 and gp41 regions were sequenced in ABI Prism 3130 Genetic Analyzer (Applied Biosystems, CA, USA) with commercial kit BigDye ® Terminator Cycle Sequencing version 3.1 (Applied Biossystems, Foster City. California, USA). Initiators of the second stage of PCR nested were used for sequencing. Results: seven patients had a higher response to T20 for 12 months, 4 for at least 6 months and two did not respond between 6 and 12 months. The average age was 44.92 ± 5.39, and 46.16% female and 53.84% male. Of the 13 patients analyzed, 12 belong to subtype B and 1 to F1. Eight patients had the coreceptor R5 and five the X4. There were no mutations at positions 36 to 45 of HR1 in 12 months. We acquired the N42S, which is responsible for decreased susceptibility to T20. From a total of 59 amino acids analyzed in HR1, we observed 18.64% of change and in HR2 region, 38.88%. In HR1 within 8 months or more compared to the baseline, we found no changes in positions 36 to 45. From a total of 59 amino acids analyzed, we observed 15.25% of change and in HR2, 36.11%. We checked the E137K without the presence of N43D, which caused resistance. We checked the S138T mutation as the primary persistence of 12 months without the N43D and the lack of response to treatment. On the count of CD4 + T cells, we found no statistical difference between the means of different groups (ANOVA - p = 0.1). The viral loads after 6 months were undetectable (<50 copies / mL) in 69.23% of patients. There was a decrease in viral load baseline to 6 months (p = 0.034) and for 6 months and 8 months or more. The use of the drug had significant interference when compared to baseline and 6 months (p = 0.004) and between baseline and 12 months (p = 0.022), but between 6 and 12 months the interference was not significant, showing that after 6 months it remains sustainable. Among the variables, responders and R5 genotype, there were high value of similarity (76.98). There was a grouping between the two samples of responders with X4 genotype with mutations 306, 311 and 320 which characterize it. Discussion: regarding tropism, 38.47% of the patients studied had correceptor X4 after a few years of infection and multiple HAART regimens. This result shows agreement with the literature, which describes an average rate of 50%. The N42S, observed in our study is associated with decreased drug susceptibility. However, only one patient did not respond to treatment, without increase of his immune. Conclusion: the tropism of HIV-1 in the majority of people who responded to the T20 is strongly associated with the R5, which causes rapid progression. The presence of S138T was sufficient for the lack of response to T20, but no association with N43D. It was clearly shown SVR and immune recovery with the T20, but that was not enough to remove these people from the risk of contracting opportunistic diseases. Our study showed the importance of starting the rescue when the count of CD4 + T cells are above 200 cells/mm3. / TEDE / BV UNIFESP: Teses e dissertações

La disponibilité des antirétroviraux,ARV pour traiter les patients infectés par le VIH a été l’un des plus grands défis de santé publique ces dernières années. Actuellement, de nouveaux ARV sont en cours de développement ou déjà disponibles, comme les inhibiteurs non-nucléosidiques de la transcriptase inverse de deuxième génération, les inhibiteurs d'intégrase et les inhibiteurs d’attachement (inhibiteurs d'entrée du virus dans la cellule). Cependant, le succès à long terme de ces traitements se heurte à plusieurs problèmes, dont l’émergence de souches résistantes du VIH aux ARV. Alors que les connaissances portent essentiellement sur le VIH-1 prévalent dans les pays du Nord, le sous-type B, la majorité des infections mondiales à VIH est causée par les sous-types non B, majoritairement présents dans les pays du Sud. L’accès aux ARV étant de plus en plus important dans les pays du Sud, il est donc important d’étudier les bases moléculaires de la résistance des VIH-1 de sous-type non-B. Dans une première partie de cette thèse, nous avons évalué la résistance primaire chez des patients infectés par le VIH dans deux contextes différents de prise en charge, au Sud (Mali) et au Nord (Hôpital de la Pitié-Salpêtrière, France). Les résultats montrent une augmentation de la résistance primaire au cours du temps au Mali, probablement liée à un accès élargi aux ARV. Nous avons également observé une fréquence de mutations de résistance plus élevée chez les virus de sous-type non-B versus sous-type B. Dans la seconde partie de ce travail, nous avons caractérisé les profils de résistance génotypique chez des patients sous traitement de première ligne depuis au moins trois ans au Mali. Nous rapportons un niveau élevé de résistance aux inhibiteurs non-nucléosidiques de la transcriptase inverse de première et de seconde génération. Enfin, dans la troisième partie de cette thèse nous nous sommes intéressés à l’étude du polymorphisme au niveau des codons nucléotidiques codant pour les acides aminés impliqués dans la résistance aux nouvelles molécules. Nous avons d’abord comparé la barrière génétique à la résistance entre les sous-types B et CRF02_AG pour la rilpivirine et l’étravirine, inhibiteurs de la transcriptase inverse de deuxième génération. Puis, nous avons comparé la barrière génétique entre le sous-type B et différents sous-types non B (C, D, CRF02_AG et CRF01_AE) pour l’inhibiteur d’attachement, BMS-626529.Les résultats montrent que la barrière génétique des sous-types non B est proche de celle du sous type B pour ces nouvelles molécules. / The availability of antiretroviral therapies, ART to treat HIVinfected patients has been one of the greatest public health concerns in the recent years. Currently, new ART are under development or already available, such as non-nucleoside reverse transcriptase second-generation,integrase inhibitors and attachment inhibitors (inhibitors of viral entry into the cell). However, the long-term success of these treatments faces several challenges, including the emergence of resistances to ART. Also, while most available data being focused on the subtype B of HIV-1 (themost prevalent in northern countries), the majority of HIVcases worldwide are caused by the non-B subtypes, mostly in the South. Access to ARTin this part of the World is significantly and regularly increasing, and therefore highlights again the need to study the molecular basis of resistance of that subtype non-B of HIV-1.In the first part of this thesis, we evaluated the primary resistance withHIV infected patients in two different contexts of health care, South (Mali) and the North (Pitié-Salpêtrière, France). The results show an increase in primary resistance over time in Mali, probably due to an expanded access to ART in the country. We also observed a higher frequency of resistance mutations in virus non-B subtype compared to thesubtype B. In the second part of this work, we characterized the genotypic resistance profiles in patients that received first-line treatment for at least three years in Mali. We found a high level of resistance to non-nucleoside reverse transcriptase first and second generations. Finally, in the third part of this thesis we were interested in studying the nucleotide polymorphism of codons encodingamino acids involved in new drugsresistances. We first compared the genetic barrier to resistance between subtypes B and CRF02_AG for rilpivirine and etravirine, inhibitors of reverse transcriptase second generation. Then, we compared the genetic barrier between subtype B and different non-B subtypes (C, D, CRF01_AE and CRF02_AG) for the attachment inhibitor, BMS-626529. The results show that the genetic barrier of non-B subtypes is similar to those of subtype B for these new molecules.

Submitted by Rosina Valeria Lanzellotti Mattiussi Teixeira (rosina.teixeira@unisantos.br) on 2015-05-05T13:17:36Z No. of bitstreams: 1 Wellington Dias.pdf: 2827942 bytes, checksum: 17fd5915b3792de99e3b58f162440777 (MD5) / Made available in DSpace on 2015-05-05T13:17:36Z (GMT). No. of bitstreams: 1 Wellington Dias.pdf: 2827942 bytes, checksum: 17fd5915b3792de99e3b58f162440777 (MD5) Previous issue date: 2013-04-17 / Introduction: The use of viral genotyping as a tool for the treatment of HIV positive patients is relevant to review health policies concerning the dispensing of antiretrovirals and contribute to mapping the frequency of HIV virus subtypes in our region. Objective: To identify and characterize the genetic diversity and primary resistance to HIV-1 antiretroviral agents in the city of itanhaem in patients at early stage of treatment . Methods: Cross-sectional study using 50 samples. After extraction and RNA transcription Polymerase Chain Reaction was performed with primers (K1, K2, DP10, F2) which produced fragments of 1200 bp, identified in agarose gel. After purification of PCR products, the fragments were subjected to sequencing reaction. Genetic diversity was analyzed throuh the comparison to the Stanford HIV drug resistance database . Qualitative variables were presented as absolute and relative values. Quantitative variables were presented through their values of central tendency and dispersion. Results: Among the 50 samples, 25 were amplified for analysis of genetic diversity, obtaining results in 13 (52%) female patients and 12 (48%) male patients. Eleven patients (44%) presented mutation in reverse transcriptase with high-level resistance to efavirez ripivirine and 7 patients (28%) presented mutations in the protease with low resistance. Four (16%) recombinant subtypes F / B were found. Conclusion: In the city of Itanhém 52% of the analyzed samples presented acquired resistance. The predominant primary resistance was to NNRTI (11 patients, representing 44%) group; One patient (4%) was found resistant to NRTI group. One patient (4%) was resistant to IP 1 group and one patient (4%) presented resistance to both NRTI and NNRTI groups. We conclude that health policies shoud be implemented considerinG genotyping as a tool to be be offered regionally, respecting the epidemiologic aspects of the disease in each city. A major 10 research concerning the most common subtype of the virus shoud be taken in our region, in order to determine the most effective drugs for antiretroviral therapy. / Introdução: A utilização da genotipagem viral como instrumento para o tratamento dos pacientes HIV positivo é relevante para rever as políticas de saúde concernentes a dispensação de antiretrovirais e contribuir para o mapeamento da freqüência do subtipo do vírus do HIV na região. Objetivo: Identificar e caracterizar a diversidade genética e a resistência primária do HIV-1 aos agentes antirretrovirais no município de Itanhaém, em pacientes em início de tratamento. Método: Estudo Transversal utilizando 50 amostras. Após a extração e trancrição do RNA foi realizada a Reação em Cadeia da Polimerase em primers(K1, K2, DP10, F2) que produziu fragmentos de 1200 pares de bases, identificados no gel de agarose. Apos a purificação dos produtos da PCR, os fragmentos foram submetidos à reação de seqüenciamento. A diversidade genética foi analisada no banco de dados HIV drug resistance database (Stanford). As variáveis qualitativas foram apresentadas através de valores absolutos e relativos. As variáveis quantitativas foram apresentadas através dos seus valores de tendência central e de dispersão. Resultados: Das 50 amostras, 25 foram amplificados para analise da diversidade genética, 13(52%) feminino e12(48%) masculino. Sendo 11(44%) identificadas com mutação na transcriptase reversa com alto nível de resistência ao efavirez ripivirine 7(28%) apresentado mutações na protease com baixo nível de resistência. Foram encontrados 4(16%) recombinantes do subtipos F/B. Conclusão: A resistência adquirida no município de Itanhaém foi de 52 % nas amostras analisadas. A resistência primaria predominante foi aos ITRNN- 11 (44 %); ITRN -1 (4 %) e aos IP -1 (4 %) e 1 (4 %) caso de resistência a dois grupos ITRNN e ITRN . Em vista disto as políticas de saúde devem considerar que a genotipagem 8 é um instrumento que deve ser oferecido regionalmente conforme o perfil epidemiológico da doença de cada município, possibilitando o estudo do subtipo do vírus mais freqüente na região afim de utilizar as drogas mais eficientes para a terapia antirretroviral.

Made available in DSpace on 2014-07-29T15:30:35Z (GMT). No. of bitstreams: 1 Dissertacao Bruna Coelho Carvalho.pdf: 623511 bytes, checksum: 1b8b7e76b1e1a914d834251448a49ea4 (MD5) Previous issue date: 2011-02-21 / Regional differences in the molecular epidemiology of HIV-1 have been reported in Brazil, where there is scarce publication about the epidemic in north region. Despite the large number of antiretroviral drugs (ARV) belonging to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) and protease inhibitors (PI) classes, the selection of resistance-associated mutations may compromise the therapeutic efficacy. Primary resistance mutations, present prior to ARV use, occur mainly due to the transmission of resistant virus. This study describes the prevalence and profile of primary resistance mutations to ARV and the molecular epidemiology of HIV-1 in ARV naive patients from Tocantins State. For HIV-1 genotyping of the entire protease gene (PR) and 750 bp of reverse transcriptase (RT) gene, plasma RNA was extracted, reverse transcribed into cDNA) and used as the target for nested polymerase chain reaction (K1/K2 and F2/DP10 primers) and fragments were sequenced (kit DYEnamic ET Dye Terminator, GE Healthcare; ABI Prism 3130). The sequences were edited by the Staden Package software. Primary drug resistance was analyzed using the Calibrated Population Resistance (CPR) tool employing the Stanford Surveillance Drug Resistance Mutation (SDRM). The susceptibility profile of ARV mutations was analyzed by Stanford HIV Drug Resistance Database (hivdb.stanford.edu). HIV-1 genetic subtypes were identified by REGA HIV-1 and SIMPLOT softwares and by phylogenetic inference. Naïve patients (n=52) were recruited in LACEN/Palmas/TO between 2008-2010. The majority of investigated patients (59.6%) were males and 73.1% reported heterosexual exposure. Primary mutations that confer resistance to ARV were identified in 11.5% (06/52) of the isolates: BRTO08-43: M41L, L210W, T215D (NRTI); BRTO02-83: Y181C (NNRTI); BRTO13-83: D67G, K219E (NRTI); BRTO20-83: V108I, Y181C (NNRTI); BRTO02-66: M46L (PI); e BRTO13-66: V90I, K103N (NNRTI). Isolates with concordant subtypes in PR/RT regions represented 86.5% (45/52): subtype BPRBRT=78.8% (41/52), subtype CPRCRT=5.8% (3/52), subtype F1PRF1RT=1.9%. Isolates with discordant subtypes in PR and RT genes indicating intersubtype recombination represented 13.5% (07/52): BPRF1TR=1.9% (01/52); BPRBF1TR=7.7% (04/52) e CPRCF1TR=3.9% (02/52). Our study among naive patients from Tocantins State describes moderate prevalence of primary resistance to ARV, the predominance of subtype B that co-circulates with subtype C and a significant number of B/F1 and C/F1 recombinant forms. These results indicate the transmission of ARV resistant HIV-1 isolates among patients from a small inland city in north Brazil, where the epidemic is more recent. In this context, it is important to monitor the prevalence of primary drug resistance in order to assess the need and the cost-benefit of the implementation of pre-treatment genotipic tests aiming to optimize the choice of the ARV regimen among naive patients from Tocantins State. / Diferenças regionais na epidemiologia molecular do HIV-1 têm sido descritas no Brasil e publicações sobre a epidemia da região Norte do país são restritas. Apesar do grande número de drogas antirretrovirais (ARV) das classes inibidores nucleosídicos e não-nucleosídicos (INTR e INNTR) da transcriptase reversa e inibidores da protease (IP), mutações associadas à resistência podem comprometer a eficácia terapêutica. Mutações de resistência primária presentes antes do uso de ARV ocorrem principalmente por transmissão de vírus resistentes. Este estudo descreve a prevalência e o perfil de mutações de resistência primária aos ARVs e subtipos do HIV-1 identificados em pacientes virgens de tratamento do estado do Tocantins. A genotipagem do gene completo da protease (PR) e 750pb da transcriptase reversa (TR) do HIV-1 foi feita a partir de RNA plasmático e incluiu retrotranscrição, nested PCR (primers K1/K2 e F2/DP10) e sequenciamento (kit DYEnamic ET Dye Terminator, GE Healthcare; ABI Prism 3130). As sequências foram editadas pelo software Staden Package. A resistência primária foi analisada pela ferramenta de Calibração da População com Resistência (CPR), empregando a ferramenta do Stanford Surveillance Drug Resistance Mutation (SDRM). O perfil de susceptibilidade dos isolados com mutação de resistência aos ARVs foi analisado pelo Stanford HIV Drug Resistance Database (hivdb.stanford.edu). Os subtipos genéticos do HIV-1 foram identificados pelos softwares REGA HIV-1, SIMPLOT e por inferência filogenética. Pacientes virgens de tratamento (n=52) foram recrutados no LACEN/Palmas/TO entre 2008-2010. A maioria dos pacientes estudados (59,6%) era do sexo masculino e 73,1% referiu exposição heterossexual. Mutações que conferem resistência primária aos ARVs foram identificadas em 11,5% (06/52) dos isolados: BRTO08-43: M41L, L210W, T215D (INTR); BRTO02-83: Y181C (INNTR); BRTO13-83: D67G, K219E (INTR); BRTO20-83: V108I, Y181C (INNTR); BRTO02-66: M46L (IP); e BRTO13-66: V90I, K103N (INNTR). Isolados com subtipos concordantes nas regiões PR/TR representaram 86,5% (45/52): subtipo BPRBTR=78,8% (41/52), subtipo CPRCTR=5,8% (03/52), subtipo F1PRF1TR=1,9%. Isolados com subtipos discordantes em PR e TR indicando recombinação intersubtipo representaram 13,5% (07/52): BPRF1TR=1,9% (01/52); BPRBF1TR=7,7% (04/52) e CPRCF1TR=3,9% (02/52). Nosso estudo em pacientes virgens de tratamento do estado do Tocantins identificou prevalência moderada de resistência primária aos ARVs, predomínio do subtipo B que co-circular com o subtipo C e um percentual significativo de formas recombinantes intersubtipos B/F1 e C/F1. Estes dados indicam a transmissão do HIV-1 resistentes a ARVs em pequenos centros urbanos no interior do Brasil, onde a epidemia é mais recente. Neste contexto, o monitoramento da prevalência de resistência primária é importante para avaliar a necessidade e custo-benefício da implantação do teste de genotipagem pré-ARV para otimizar a escolha do esquema ARV em pacientes do estado do Tocantins.

Une transition épithélio-mésenchymateuse (TEM) et une expression élevée de la sphingosine kinase 1 (SPHK1) sont souvent observées dans les cancers. Notre étude du génome et du transcriptome d'adénocarcinomes pulmonaires (AP) montre que l'expression élevée de SPHK1 est en rapport, d'une part, avec des gains de la région incluant le locus SPHK1 et, d'autre part, avec une signature d'expression génique de TEM dans des tumeurs invasives. L'expression de SPHK1 est restreinte aux cellules tumorales. La surexpression de SPHK1 dans des cellules d'AP et l'exposition à son produit, la sphingosine-1-phosphate (S1P), entraînent une TEM, de manière réversible pour la S1P. La surexpression de SPHK1 active aussi NF-kB. La surexpression du facteur anti-apoptotique FLIP active NF-kB, induit une TEM et augmente l'expression de SPHK1, suggérant une boucle d'amplification entre NF-kB et SPHK1. Une TEM et la surexpression de FLIP ont été impliquées dans la résistance primaire aux inhibiteurs pharmacologiques de l'EGFR (EGFR TKI). Nous montrons que la surexpression de SPHK1 dans des cellules A549 diminue modestement la sensibilité au gefitinib, alors que l'inhibition de SPHK1 ou la déplétion du sérum en S1P l'augmentent modestement. L'invalidation de SPHK1 entraîne l'apoptose d'A549 y compris quand FLIP est surexprimé. L'activation et le maintien d'une TEM sont généralement attribués à des signaux contextuels du stroma. Cette thèse montre que les cellules tumorales elles-mêmes favorisent la surexpression de SPHK1 qui peut induire une TEM de façon autonome. De plus, la surexpression de FLIP impliquée dans la résistance aux EGFR TKI, n'empêche pas l'apoptose induite par l'invalidation de SPHK1. / Epithelial-mesenchymal transition (EMT) and sphingosine kinase 1 (SPHK1) high expression are often seen in cancers. Our study of genomic and gene expression data in pulmonary adenocarcinomas (AP) shows that SPHK1 high expression correlates with both gains in the region encompassing the SPHK1 locus, and an EMT gene expression signature in invasive tumors. SPHK1 expression is restricted to tumors cells. SPHK1 overexpression in AP cells, as well as exposure to its productsphingosine-1-phosphate (S1P),induce an EMT -in a reversible manner for S1P. SPHK1 overexpression also activates NF-kB. Overexpression of FLIP – an antiapoptotic factor - activates NF-kB, induces an EMT, and increases SPHK1 expression, suggesting an amplification loop between NF-kB and SPHK1. EMT and FLIP overexpression are known to favor primary resistance to EGFR pharmacological inhibitors (EGFR TKI). We show that SPHK1 overexpression in A549 cells slightly decreases cell sensitivity to gefitinib, while pharmacologic inhibition of SPHK1 or serum S1P depletionincrease it. Downregulation of SPHK1 expression induces apoptosis of A549 cells even when FLIP is overexpressed. Activation and maintenance of EMT are generally attributed to contextual signals from the stroma. Here, we show that tumor cells themselves favor SPHK1 overexpression, which can led to EMT in cell-autonomous manner. In addition, FLIP overexpression which is implicated in EGFR TKI resistance, cannot prevent apoptosis that is induced by SPHK1 invalidation.

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.

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.